Therapeutic microbiota for the treatment and/or prevention of food allergy

ABSTRACT

Disclosed are methods and compositions for the prevention and treatment of food allergy. In particular, described herein are microbial consortia, including minimal microbial consortia, that can prevent and/or cure food allergy. In certain embodiments, the consortia comprise certain members of the taxa Clostridiales and/or Bacteroidetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation Application of InternationalApplication No. PCT/US2016/060353 filed Nov. 3, 2016, which designatesthe U.S. and which claims benefit under 35 U.S.C. § 119(e) of the U.S.Provisional Application No. 62/250,277 filed Nov. 3, 2015, the contentsof each of which are incorporated herein by reference in theirentireties.

GOVERNMENT SUPPORT

This invention was made with Government Support under Grant Nos.1R56A111798-01 and P30 DK056338 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure relates to the treatment and/or prevention offood allergy.

BACKGROUND

Food allergies are a growing public health problem in both developed andrapidly developing countries and affects large numbers of children andadults. The incidence of food allergy has increased dramatically in thelast few decades. This increase can be associated with sensitization tomultiple foods in up to 50% of subjects. Growing evidence indicates thatthe microbial flora is a key environmental influence in programming oraltolerance.

SUMMARY

Provided herein are methods and compositions for the treatment and/orprevention of food allergy, based in part, on the discovery that alteredintestinal microbiota (e.g., from antibiotic treatments, C-sectionbirths, diet etc.) can promote food allergy while some combinations ofmicrobes can prevent and/or cure food allergies.

Accordingly, provided herein in one aspect is a pharmaceuticalcomposition comprising: (i) a preparation comprising a minimal microbialconsortium consisting essentially of four to eleven strains of viablegut bacteria, in an amount sufficient to treat or prevent a food allergywhen administered to an individual in need thereof, and (ii) apharmaceutically acceptable carrier.

Another aspect provided herein relates to a pharmaceutical compositioncomprising a minimal microbial consortium of culturable species, and apharmaceutically acceptable carrier, wherein the consortium is comprisedin at least four of the preparations selected from the group consistingof: (i) a preparation of a viable, culturable, anaerobic gut bacterialstrain(s) that expresses exopolysaccharide, lipoteichoic acid (LTA),lipopolysaccharide (LPS) or other microbial adjuvant molecules thatpromote the development of regulatory T cells (Treg); (ii) a preparationof a viable, culturable, anaerobic gut bacterial strain(s) that producesbutyrate and/or propionate fermentation products via fermentation ofcarbohydrates and other carbon sources in the gut lumen; (iii) apreparation of one or more viable, culturable, anaerobic gut bacterialstrains that alone or in combination performs the full complement ofbile acid transformations; (iv) a preparation of a viable, culturable,anaerobic gut bacterial strain that produces compounds capable ofstimulating the aryl hydrocarbon receptor (AhR) receptor pathway in gutepithelial cells, antigen presenting cells and/or T cells to stimulatedevelopment of regulatory T cell responses; (v) a preparation of aviable, culturable, anaerobic gut bacterial strain(s) that producescompounds capable of stimulating the pregnane X receptor with beneficialeffects upon gut barrier function and/or development of regulatory Tcell responses; (vi) a preparation of a viable, culturable, anaerobicgut bacterial strain(s) that produces compounds capable of stimulatingthe RORgamma (RAR-related orphan receptor gamma) pathways to stimulatedevelopment of regulatory T cell responses via direct stimulation orRORgamma-activated pathways in gut antigen presenting cells and/orepithelial cells that then stimulate regulatory T cell responses; (vii)a preparation of viable, culturable, anaerobic gut bacterial strain(s)that stimulates host production of mucins and complex glycoconjugatesthat improve gut barrier function and colonization by protectivecommensal species; (viii) a preparation of a viable, culturable,anaerobic gut bacterial strain(s) that alters the gut luminalenvironment to reduce the deleterious activities of dysbiotic speciespromoting development of unhealthy allergic T cell responses to foodantigens; (ix) a preparation of a viable, culturable, anaerobic gutbacterial strain(s) that alters the gut luminal environment to promoteimproved colonization by other members of the administered consortiumfor any of the above stated effects, and/or colonization by existingbeneficial species in the patients underlying microbiota; (x) apreparation of a viable, culturable, anaerobic gut bacterial strain(s)that promotes the colonization or growth of a bacterial strain in apreparation of (i)-(ix) above, in vivo.

In one embodiment of this aspect and all other aspects described herein,the viable, culturable, anaerobic gut bacterial strain(s) that expressesexopolysaccharide that promotes the development of regulatory T cells(Treg) is selected from the group consisting of: Eubacterium rectale,Clostridium ramosum, Butyrovibrio crossatus, Roseburia intestinalis,Clostridium scindens, Clostridium hylemonae, Hungatella hathawayi,Clostridium symbiosum, Faecalibacterium prausnitzii, Subdoligranulumvariabile, Bacteroides thetaiotaomicron, Bacteroides fragilis,Bacteroides ovatus, Parabacetroides goldsteinii, Parabacteroides merdae,Parabacteroides distasonis, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the viable, culturable, anaerobic gut bacterial strain(s) thatproduces butyrate, propionate and/or succinate fermentation products viafermentation of carbohydrates in the gut lumen is selected from thegroup consisting of: Bacteroides fragilis, Bacteroides thetaiotaomicron,Bacteroides ovatus, Clostridium sardiniensis, Clostridium hiranonsis,Facealibacterium prausnitzii, Butyrovibrio spp., Eubacterium rectale andRoseburia intestinalis

In another embodiment of this aspect and all other aspects providedherein, the one or more viable, culturable, anaerobic gut bacterialstrains that alone or in combination performs the full complement ofbile acid transformations is Clostridium scindens, Clostridiumhiranonsis, Clostridium sardiniensis and/or Bacteroides spp.

In another embodiment of this aspect and all other aspects providedherein, the viable, culturable, anaerobic gut bacterial strain(s) thatproduces aryl hydrocarbon receptor agonists sufficient to stimulate hostaryl hydrocarbon receptor pathways comprises at least one geneassociated with the synthesis of tryptophan, tyrosine or the synthesisof quinone molecules.

In another embodiment of this aspect and all other aspects providedherein, the viable, culturable, anaerobic gut bacterial strain(s) thatpromotes the colonization or growth of a bacterial strain in apreparation is Bacteroides thetaiotaomicron, or Bacteroides fragilis.

In another embodiment of this aspect and all other aspects providedherein, the viable, culturable, anaerobic gut bacterial strain(s) thatproduces compounds endogenously or by metabolizing exogenous precursors,that is capable of stimulating the pregnane X receptor with beneficialeffects upon gut barrier function and/or development of regulatory Tcell responses is a strain that expresses desmolase and/orhydroxysteroid dehydrogenase. In this context, exogenous precursorsinclude not just dietary compounds or factors, but also factors producedby the host and excreted into the gut which are then acted upon by oneor more members of a microbial consortium as described herein.

In another embodiment of this aspect and all other aspects providedherein, the viable, culturable, anaerobic gut bacterial strain(s) thatproduces compounds endogenously or by metabolizing exogenous precursors,that is capable of stimulating the RORgamma (RAR-related orphan receptorgamma) pathways to stimulate development of regulatory T cell responsesis a strain that expresses at least one cholesterol reductase or anotherenzyme capable of metabolizing sterol compounds.

In another embodiment of this aspect and all other aspects providedherein, the viable, culturable, anaerobic gut bacterial strain(s) thatis capable of stimulating host mucins and complex glycoconjugates thatimprove gut barrier function and colonization by protective commensalspecies is Bacteroides thetaiotaomicron or Bacteroides fragilis.

In another embodiment of this aspect and all other aspects providedherein, the viable, culturable, anaerobic gut bacterial strain(s) thatalter the gut luminal environment to reduce the deleterious activitiesof dysbiotic species that contribute to development of pathogenicallergic T cell responses to food antigens is Bacteroidesthetaiotaomicron, Bacteroides fragilis or another Bacteroides spp. Anexamples includes alterations of the gut environment from Bacteroidesspecies that reduces the biomass of dysbiotic species in theEnterobacteriaceae or Desulfonovibriaceae and/or prevent full expressionof dysbiotic biochemical or microbiologic activities expressed by thesespecies.

In another embodiment of this aspect and all other aspects providedherein, the viable, culturable, anaerobic gut bacterial strain(s) alterthe gut luminal environment to promote improved colonization by othermembers of the administered consortium for any of the above statedeffects, and/or colonization by existing beneficial species in thepatients underlying microbiota is Bacteroides thetaiotaomicron,Bacteroides fragilis, or Bacteroides spp. In another embodiment of thisaspect and all other aspects provided herein, the beneficial speciescomprises Clostridium spp (e.g., Clostridium ramosum, Clostridiumscindens, Clostridium hiranonsis, Clostridium bifermentans, Clostridiumleptum, Clostridium sardiniensis, Clostridium hathewayi, Clostridiumnexile, Clostridium hylemonae, Clostridium glycyrrhizinilyticum,Clostridium lavalense, Clostridium fimetarium, Clostridium symbiosum,Clostridium sporosphaeroides etc.) or another non-pathogenic commensalstrain.

In another embodiment of this aspect and all other aspects providedherein, the pharmaceutically acceptable carrier comprises an entericcoating composition that encapsulates the minimal microbial consortium.

In another embodiment of this aspect and all other aspects providedherein, the pharmaceutical composition is formulated to deliver theviable bacteria to the small intestine.

In another embodiment of this aspect and all other aspects providedherein, the enteric-coating composition is in the form of a capsule,gel, pastille, tablet or pill.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable, culturable, anaerobic gut bacteria arehuman gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable, culturable, anaerobic gut bacteria areselected from the group consisting of: Clostridium ramosum, Clostridiumscindens, Clostridium hiranonsis, Clostridium bifermentans, Clostridiumleptum, Clostridium sardiniensis, Bacteroides fragilis, Bacteroidesthetaiotaomicron, Bacteroides ovatus, Parabacteroides goldsteinii,Prevotella tannerae, Clostridum hathewayi, Clostridum nexile,Clostridium hylemonae, Clostridium glycyrrhizinilyticum, Clostridiumscindens, Clostridium lavalense, Clostridum fimetarium, Clostridiumsymbiosum, Clostridium sporosphaeroides, Dialister proprionicfaceins,Dialister succinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable, culturable, anaerobic gut bacteria areselected from the group consisting of: Clostridium ramosum, C. scindens,C. hiranonsis, C. bifermentans, C. leptum, C. sardiniensis, Bacteroidesfragilis, B. thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii,and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable, culturable, anaerobic gut bacteria areselected from the group consisting of: (i) Clostridium ramosum, C.scindens, C. hiranonsis, C. bifermentans, C. leptum, and C.sardiniensis, or (ii) Bacteroides fragilis, B. thetaiotaomicron, B.ovatus, Parabacteroides goldsteinii, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable, culturable, anaerobic gut bacteria arepresent in substantially equal biomass.

In another embodiment of this aspect and all other aspects providedherein, the composition is formulated to deliver a dose of at least1×10⁹ colony forming units (CFUs).

In another embodiment of this aspect and all other aspects providedherein, the composition is formulated to deliver at least 1×10⁹ CFUs inless than 30 capsules per one time dose.

In another embodiment of this aspect and all other aspects providedherein, the composition is frozen for storage.

In another embodiment of this aspect and all other aspects providedherein, the strain(s) of viable culturable, anaerobic gut bacteria areencapsulated under anaerobic conditions.

In another embodiment of this aspect and all other aspects providedherein, the strain(s) of viable culturable, anaerobic gut bacteria arelyophilized under anaerobic conditions.

In another embodiment of this aspect and all other aspects providedherein, anaerobic conditions comprise one or more of the following: (i)oxygen impermeable capsules, (ii) addition of reducing agents includingN-acetylcysteine, cysteine, or methylene blue to the composition, and(iii) use of spores for organisms that sporulate.

In another embodiment of this aspect and all other aspects providedherein, the consortium comprises at least one bacterial straincomprising a 16S rDNA sequence at least 97% identical to a 16S rDNAsequence present in a reference strain operational taxonomic unit, thereference strain selected from the group consisting of: Clostridiumramosum, C. scindens, C. hiranonsis, C. bifermentans, C. leptum, and C.sardiniensis, or wherein the consortium comprises at least one bacterialstrain comprising a 16S rDNA sequence at least 97% identical to a 16SrDNA sequence present in a reference strain operational taxonomic unit,the reference strain selected from the group consisting of: Bacteroidesfragilis, B. thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii,and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the consortium does not comprise any of the Species Escherichiacoli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae,Bilophila wadsworthia, Alistipes onderdonkii, Desulfovibrio species,Lactobacillus johnsoni, Parasutterella excrementihominis.

In another embodiment of this aspect and all other aspects providedherein, the consortium does not comprise bacteria of the GeneraBilophila, Enterobacter, Escherichia, Klebsiella, Proteus, Alistipes,Blautia, Desulfovibrio, or Parasutterella.

In another embodiment of this aspect and all other aspects providedherein, the consortium does not comprise bacteria of the FamiliesDesulfovibrionaceae, Enterobacteriaceae, Rikenellaceae, andSutterellaceae.

In another embodiment of this aspect and all other aspects providedherein, the consortium does not comprise bacteria of the FamiliesLactobacillaceae, or Enterbacteriaceae.

In another embodiment of this aspect and all other aspects providedherein, the consortium does not comprise bacteria of the OrderBurkholdales, Desulfovibrionales, or Enterobacteriales.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least five strains of viablenon-pathogenic gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises up to eleven strains of viablenon-pathogenic gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum, and C. sardiniensis.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises Bacteroides fragilis, B.thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii, and Prevotellatannerae.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises: Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum, C. sardiniensis, Bacteroidesfragilis, B. thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii,and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the bacterial strains in the composition consists essentially ofClostridium ramosum, C. scindens, C. hiranonsis, C. bifermentans, C.leptum, and C. sardiniensis.

In another embodiment of this aspect and all other aspects providedherein, the microbial consortium consists essentially of Bacteroidesfragilis, B. thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii,and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the microbial consortium consists essentially of: Clostridiumramosum, C. scindens, C. hiranonsis, C. bifermentans, C. leptum, C.sardiniensis, Bacteroides fragilis, B. thetaiotaomicron, B. ovatus,Parabacteroides goldsteinii, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the enteric-coating comprises a polymer, nanoparticle, fattyacid, shellac, or a plant fiber.

In another embodiment of this aspect and all other aspects providedherein, the composition further comprises a pre-biotic composition.

In another embodiment of this aspect and all other aspects providedherein, the composition is encapsulated, a reconstituted lyophilisate, afood item, or is formulated as a liquid, gel, fluid-gel, ornanoparticles in a liquid.

Another aspect provided herein relates to a pharmaceutical compositioncomprising: (i) a preparation comprising at least four strains ofviable, anaerobic, culturable gut bacteria selected from the groupconsisting of: Clostridium ramosum, Clostridium scindens, Clostridiumhiranonsis, Clostridium bifermentans, Clostridium leptum, Clostridiumsardiniensis, Bacteroides fragilis, Bacteroides thetaiotaomicron,Bacteroides ovatus, Parabacteroides goldsteinii, Prevotella tannerae,Clostridum hathewayi, Clostridum nexile, Clostridium hylemonae,Clostridium glycyrrhizinilyticum, Clostridium scindens, Clostridiumlavalense, Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti, in an amount sufficientto treat or prevent a food allergy when administered to an individual inneed thereof, and (ii) a pharmaceutically acceptable carrier.

In one embodiment of this aspect and all other aspects provided herein,the composition comprises not more than forty strains of viable,anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than thirty strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than twenty strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than fifteen strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than eleven strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least five strains of viable,anaerobic, culturable gut bacteria selected from the group consistingof: Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis,Clostridium bifermentans, Clostridium leptum, Clostridium sardiniensis,Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus,Parabacteroides goldsteinii, Prevotella tannerae, Clostridum hathewayi,Clostridum nexile, Clostridium hylemonae, Clostridiumglycyrrhizinilyticum, Clostridium scindens, Clostridium lavalense,Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least six strains of viable,anaerobic, culturable gut bacteria selected from the group consistingof: Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis,Clostridium bifermentans, Clostridium leptum, Clostridium sardiniensis,Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus,Parabacteroides goldsteinii, Prevotella tannerae, Clostridum hathewayi,Clostridum nexile, Clostridium hylemonae, Clostridiumglycyrrhizinilyticum, Clostridium scindens, Clostridium lavalense,Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least seven strains of viable,anaerobic, culturable gut bacteria selected from the group consistingof: Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis,Clostridium bifermentans, Clostridium leptum, Clostridium sardiniensis,Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus,Parabacteroides goldsteinii, Prevotella tannerae, Clostridum hathewayi,Clostridum nexile, Clostridium hylemonae, Clostridiumglycyrrhizinilyticum, Clostridium scindens, Clostridium lavalense,Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least eight strains of viable,anaerobic, culturable gut bacteria selected from the group consistingof: Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis,Clostridium bifermentans, Clostridium leptum, Clostridium sardiniensis,Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus,Parabacteroides goldsteinii, Prevotella tannerae, Clostridum hathewayi,Clostridum nexile, Clostridium hylemonae, Clostridiumglycyrrhizinilyticum, Clostridium scindens, Clostridium lavalense,Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least nine strains of viable,anaerobic, culturable gut bacteria selected from the group consistingof: Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis,Clostridium bifermentans, Clostridium leptum, Clostridium sardiniensis,Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus,Parabacteroides goldsteinii, Prevotella tannerae, Clostridum hathewayi,Clostridum nexile, Clostridium hylemonae, Clostridiumglycyrrhizinilyticum, Clostridium scindens, Clostridium lavalense,Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least ten strains of viable,anaerobic, culturable gut bacteria selected from the group consistingof: Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis,Clostridium bifermentans, Clostridium leptum, Clostridium sardiniensis,Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus,Parabacteroides goldsteinii, Prevotella tannerae, Clostridum hathewayi,Clostridum nexile, Clostridium hylemonae, Clostridiumglycyrrhizinilyticum, Clostridium scindens, Clostridium lavalense,Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In other embodiments of this aspect and all other aspects providedherein, the composition comprises a range of 4-28, 4-35, 4-30, 4-25,4-20, 4-15, 4-12, 4-11, 4-10, 4-9, 4-8, 4-6, 35-40, 30-40, 25-40, 20-40,15-40, 12-40, 11-40, 10-40, 6-40, 5-40, 10-20, 10-30, 10-25, 15-40,15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 20-25, 30-35 strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable, anaerobic, culturable gut bacteria areselected from the group consisting of: (i) Clostridium ramosum, C.scindens, C. hiranonsis, C. bifermentans, C. leptum, and C.sardiniensis, or (ii) Bacteroides fragilis, B. thetaiotaomicron, B.ovatus, Parabacteroides goldsteinii, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises viable, anaerobic, culturable gutbacteria including each of Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum and C. sardiniensis.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises viable, anaerobic, culturable gutbacteria including each of, Bacteroides fragilis, B. thetaiotaomicron,B. ovatus, Parabacteroides goldsteinii, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises viable, anaerobic, culturable gutbacteria including each of Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum, C. sardiniensis, Bacteroidesfragilis, B. thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii,and Prevotella tannerae.

Another aspect described herein relates to a pharmaceutical compositioncomprising: at least four strains of viable, anaerobic, culturable gutbacteria comprising: at least one bacterial strain comprising a 16S rDNAsequence at least 97% identical to a 16S rDNA sequence present in areference strain operational taxonomic unit, the reference strainselected from the group consisting of: Clostridium ramosum, C. scindens,C. hiranonsis, C. bifermentans, C. leptum, and C. sardiniensis; or atleast one bacterial strain comprising a 16S rDNA sequence at least 97%identical to a 16S rDNA sequence present in a reference strainoperational taxonomic unit, the reference strain selected from the groupconsisting of: Bacteroides fragilis, B. thetaiotaomicron, B. ovatus,Parabacteroides goldsteinii, and Prevotella tannerae; wherein the atleast four strains of viable, anaerobic, culturable gut bacteria arepresent in an amount sufficient to treat or prevent a food allergy whenadministered to an individual in need thereof; and a pharmaceuticallyacceptable carrier.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than forty strains of viable,anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than thirty strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than twenty strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than fifteen strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises not more than eleven strains ofviable, anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least five strains of viable,anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least six strains of viable,anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least seven strains of viable,anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least eight strains of viable,anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least nine strains of viable,anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least ten strains of viable,anaerobic, culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises eleven strains of viable, anaerobic,culturable gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the microbial strains do not comprise any of the SpeciesEscherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobactercloacae, Bilophila wadsworthia, Alistipes onderdonkii, Desulfovibriospecies, Lactobacillus johnsoni, and Parasutterella excrementihominis.

In another embodiment of this aspect and all other aspects providedherein, the microbial strains do not comprise bacteria of the GeneraBilophila, Enterobacter, Escherichia, Klebsiella, Proteus, Alistipes,Blautia, Desulfovibrio, or Parasutterella.

In another embodiment of this aspect and all other aspects providedherein, the microbial strains do not comprise bacteria of the FamiliesDesulfovibrionaceae, Enterobacteriaceae, Rikenellaceae, andSutterellaceae.

In another embodiment of this aspect and all other aspects providedherein, the microbial strains do not comprise bacteria of the FamiliesLactobacillaceae, or Enterbacteriaceae

In another embodiment of this aspect and all other aspects providedherein, the microbial strains do not comprise bacteria of the OrderBurkholdales, Desulfovibrionales, or Enterobacteriales.

Another aspect provided herein relates to a pharmaceutical compositioncomprising a microbial consortium of culturable species, and apharmaceutically acceptable carrier, wherein the composition comprisesat least four preparations selected from the group consisting of: (i) apreparation of a viable, culturable, anaerobic gut bacterial strain (orstrains) that expresses exopolysaccharide, lipoteichoic acid (LTA),lipopolysaccharide (LPS) or other microbial adjuvant molecules thatpromote the development of regulatory T cells (Treg); (ii) a preparationof a viable, culturable, anaerobic gut bacterial strain (or strains)that produces butyrate and/or propionate fermentation products viafermentation of carbohydrates and other carbon sources in the gut lumen;(iii) a preparation of one or more viable, culturable, anaerobic gutbacterial strains that alone or in combination performs the fullcomplement of bile acid transformations; (iv) a preparation of a viable,culturable, anaerobic gut bacterial strain (or strains) that producescompounds capable of stimulating the aryl hydrocarbon receptor (AhR)receptor pathway in gut epithelial cells, antigen presenting cellsand/or T cells to stimulate development of regulatory T cell responses;(v) a preparation of a viable, culturable, anaerobic gut bacterialstrain (or strains) that produces compounds capable of stimulating thepregnane X receptor with beneficial effects upon gut barrier functionand/or development of regulatory T cell responses; (vi) a preparation ofa viable, culturable, anaerobic gut bacterial strain (or strains) thatproduces compounds capable of stimulating the RORgamma (RAR-relatedorphan receptor gamma) pathways to stimulate development of regulatory Tcell responses via direct stimulation or RORgamma-activated pathways ingut antigen presenting cells and/or epithelial cells that then stimulateregulatory T cell responses; (vii) a preparation of a viable,culturable, anaerobic gut bacterial strain (or strains) that stimulateshost production of mucins and complex glycoconjugates that improve gutbarrier function and colonization by protective commensal species;(viii) a preparation of a viable, culturable, anaerobic gut bacterialstrain (or strains) that alters the gut luminal environment to reducethe deleterious activities of dysbiotic species promoting development ofunhealthy allergic T cell responses to food antigens; (ix) a preparationof a viable, culturable, anaerobic gut bacterial strain (or strains)that alters the gut luminal environment to promote improved colonizationby other members of the administered consortium for any of the abovestated effects, and/or colonization by existing beneficial species inthe patients underlying microbiota; and (x) a preparation of a viable,culturable, anaerobic gut bacterial strain (or strains) that promotesthe colonization or growth of a bacterial strain in a preparation of(i)-(ix) above, in vivo; and wherein the composition comprises no morethan forty microbial species.

In one embodiment of this aspect and all other aspects provided herein,the composition comprises no more than thirty microbial species.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises no more than twenty microbial species.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises no more than fifteen microbialspecies.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises no more than eleven microbial species.

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least five of the preparations(i)-(x).

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least six of the preparations(i)-(x).

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least seven of the preparations(i)-(x).

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least eight of the preparations(i)-(x).

In another embodiment of this aspect and all other aspects providedherein, the composition comprises at least nine of the preparations(i)-(x).

In another embodiment of this aspect and all other aspects providedherein, the composition comprises each of the preparations (i)-(x).

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strain that expressesexopolysaccharide that promotes the development of regulatory T cells(Treg) is selected from the group consisting of: Eubacterium rectale,Clostridium ramosum, Butyrovibrio crossatus, Roseburia intestinalis,Clostridium scindens, Clostridium hylemonae, Hungatella hathawayi,Clostridium symbiosum, Faecalibacterium prausnitzii, Subdoligranulumvariabile, Bacteroides thetaiotaomicron, Bacteroides fragilis,Bacteroides ovatus, Parabacetroides goldsteinii, Parabacteroides merdae,Parabacteroides distasonis, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strain that strain that producesbutyrate, propionate and/or succinate fermentation products viafermentation of carbohydrates in the gut lumen is selected from thegroup consisting of: Bacteroides fragilis, Bacteroides thetaiotaomicron,Bacteroides ovatus, Clostridium sardiniensis, Clostridium hiranonsis,Facealibacterium prausnitzii, Butyrovibrio spp., Eubacterium rectale,and Roseburia intestinalis.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strain that alone or in combinationperforms the full complement of bile acid transformations lumen isselected from the group consisting of: Bacteroides fragilis, Bacteroidesthetaiotaomicron, Bacteroides ovatus, Clostridium sardiniensis,Clostridium hiranonsis, Facealibacterium prausnitzii, Butyrovibrio spp.,Eubacterium rectale, and Roseburia intestinalis.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strain that produces arylhydrocarbon receptor agonists sufficient to stimulate host arylhydrocarbon receptor pathways comprises at least one gene associatedwith the synthesis of tryptophan, tyrosine or the synthesis of quinonemolecules

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strain that promotes thecolonization or growth of a bacterial strain in a preparation recited inclaim 2 is Bacteroides thetaiotaomicron, or Bacteroides fragilis.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strain that produces compoundsendogenously or by metabolizing ingested precursors, that is capable ofstimulating the pregnane X receptor with beneficial effects upon gutbarrier function and/or development of regulatory T cell responses is astrain that expresses desmolase and/or hydroxysteroid dehydrogenase.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strain that produces compoundsendogenously or by metabolizing ingested precursors, that is capable ofstimulating the RORgamma (RAR-related orphan receptor gamma) pathways tostimulate development of regulatory T cell responses is a strain thatexpresses at least one cholesterol reductase or another enzyme(s)capable of metabolizing sterol compounds.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strain that is capable ofstimulating host mucins and complex glycoconjugates that improve gutbarrier function and colonization by protective commensal species isBacteroides thetaiotaomicron or Bacteroides fragilis.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strains that alter the gut luminalenvironment to reduce the deleterious activities of dysbiotic speciesthat contribute to development of pathogenic allergic T cell responsesto food antigens is Bacteroides thetaiotaomicron, Bacteroides fragilis,or Bacteroides spp. In another embodiment of this aspect and all otheraspects provided herein, the dysbiotic species comprises a species inthe Families Enterobacteriaceae or Desulfonovibriacaea.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic gut bacterial strains alter the gut luminalenvironment to promote improved colonization by other members of theadministered consortium for any of the above stated effects, and/orcolonization by existing beneficial species in the patients underlyingmicrobiota is Bacteroides thetaiotaomicron, Bacteroides fragilis, orBacteroides spp. In another embodiment of this aspect and all otheraspects provided herein, the beneficial species comprises non-pathogenicClostridia spp. and other non-pathogenic commensal strains.

In another embodiment of this aspect and all other aspects providedherein, the pharmaceutically acceptable carrier comprises an entericcoating composition that encapsulates the anaerobic gut bacterialstrains.

In another embodiment of this aspect and all other aspects providedherein, the composition is formulated to deliver the viable bacteria tothe small intestine.

In another embodiment of this aspect and all other aspects providedherein, wherein the enteric-coating composition is in the form of acapsule, gel, pastille, tablet or pill.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable, anaerobic gut bacteria are humananaerobic gut bacteria.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable anaerobic gut bacteria are selected fromthe group consisting of: Clostridium ramosum, Clostridium scindens,Clostridium hiranonsis, Clostridium bifermentans, Clostridium leptum,Clostridium sardiniensis, Bacteroides fragilis, Bacteroidesthetaiotaomicron, Bacteroides ovatus, Parabacteroides goldsteinii,Prevotella tannerae, Clostridum hathewayi, Clostridum nexile,Clostridium hylemonae, Clostridium glycyrrhizinilyticum, Clostridiumscindens, Clostridium lavalense, Clostridum fimetarium, Clostridiumsymbiosum, Clostridium sporosphaeroides, Dialister proprionicfaceins,Dialister succinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable anaerobic gut bacteria are selected fromthe group consisting of: Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum, C. sardiniensis, Bacteroidesfragilis, B. thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii,and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable anaerobic gut bacteria are selected fromthe group consisting of: (i) Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum, and C. sardiniensis, or (ii)Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, Parabacteroidesgoldsteinii, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable anaerobic gut bacteria consist essentiallyof Clostridium ramosum, C. scindens, C. hiranonsis, C. bifermentans, C.leptum, and C. sardiniensis.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable anaerobic gut bacteria consist essentiallyof Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, Parabacteroidesgoldsteinii, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable anaerobic gut bacteria consist essentiallyof Clostridium ramosum, C. scindens, C. hiranonsis, C. bifermentans, C.leptum, C. sardiniensis, Bacteroides fragilis, B. thetaiotaomicron, B.ovatus, Parabacteroides goldsteinii, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable anaerobic gut bacteria are present insubstantially equal biomass.

In another embodiment of this aspect and all other aspects providedherein, the composition is formulated to deliver a dose of at least1×10⁹ colony forming units (CFUs).

In another embodiment of this aspect and all other aspects providedherein, the composition is formulated to deliver at least 1×10⁹ CFUs inless than 30 capsules per one time dose.

In another embodiment of this aspect and all other aspects providedherein, the composition is frozen for storage.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable anaerobic gut bacteria are encapsulatedunder anaerobic conditions. In another embodiment of this aspect and allother aspects provided herein, the strains of viable anaerobic gutbacteria are lyophilized under anaerobic conditions.

In another embodiment of this aspect and all other aspects providedherein, wherein anaerobic conditions comprise one or more of thefollowing: (i) oxygen impermeable capsules, (ii) addition ofN-acetylcysteine, cysteine or methylene blue to the composition, (iii)use of spores for organisms that sporulate, and (iv) addition of areducing factor to the composition.

In another embodiment of this aspect and all other aspects providedherein, the enteric-coating comprises a polymer, nanoparticle, fattyacid, shellac, or a plant fiber.

In another embodiment of this aspect and all other aspects providedherein, the composition further comprises a pre-biotic composition.

In another embodiment of this aspect and all other aspects providedherein, the composition is encapsulated, a reconstituted lyophilisate, afood item, or is formulated as a liquid, gel, fluid-gel, ornanoparticles in a liquid.

Another aspect described herein relates to a method for preventing theonset of a food allergy in a subject, the method comprising:administering to a subject a composition as described herein, therebypreventing the onset of a food allergy in the subject.

In one embodiment of this aspect and all other aspects provided herein,the at least 4 strains of gut bacteria administered are selected fromthe group consisting of: Clostridium ramosum, Clostridium scindens,Clostridium hiranonsis, Clostridium bifermentans, Clostridium leptum,Clostridium sardiniensis, Bacteroides fragilis, Bacteroidesthetaiotaomicron, Bacteroides ovatus, Parabacteroides goldsteinii,Prevotella tannerae, Clostridum hathewayi, Clostridum nexile,Clostridium hylemonae, Clostridium glycyrrhizinilyticum, Clostridiumscindens, Clostridium lavalense, Clostridum fimetarium, Clostridiumsymbiosum, Clostridium sporosphaeroides, Dialister proprionicfaceins,Dialister succinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the strains of gut bacteria administered are selected from thegroup consisting of: Clostridium ramosum, C. scindens, C. hiranonsis, C.bifermentans, C. leptum, C. sardiniensis, Bacteroides fragilis, B.thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii, and Prevotellatannerae.

In another embodiment of this aspect and all other aspects providedherein, the strains of gut bacteria administered are selected from thegroup consisting of: (i) Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum, and C. sardiniensis, or (ii)Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, Parabacteroidesgoldsteinii, and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the composition is administered by oral administration, enema,suppository, or orogastric tube.

In another embodiment of this aspect and all other aspects providedherein, the minimal microbial consortium or viable, culturable,anaerobic gut bacterial strain(s) is/are isolated and/or purified from asubject known to be tolerant to a selected food allergen.

In another embodiment of this aspect and all other aspects providedherein, the strains of gut bacteria are cultured under anaerobicconditions.

In another embodiment of this aspect and all other aspects providedherein, the anaerobic conditions comprise one or more of the following:(i) oxygen impermeable capsules, (ii) addition of N-acetylcysteine,cysteine, methylene blue, or a reducing factor to the composition, or(iii) use of spores for organisms that sporulate.

In another embodiment of this aspect and all other aspects providedherein, the composition administered further comprises a pre-bioticcomposition.

In another embodiment of this aspect and all other aspects providedherein, the composition is enteric-coated.

In another embodiment of this aspect and all other aspects providedherein, the treatment administered prevents and/or reverses TH2programming of Tregs and other mucosal T cell populations.

In another embodiment of this aspect and all other aspects providedherein, the subject is a human subject.

In another embodiment of this aspect and all other aspects providedherein, the method further comprises a step of diagnosing the subject aslikely to develop a food allergy.

In another embodiment of this aspect and all other aspects providedherein, the method further comprising a step of testing a fecal samplefrom the subject for the presence and/or levels of the bacteria in theminimal microbial consortium or the viable, culturable, anaerobic gutbacterial strain(s).

In another embodiment of this aspect and all other aspects providedherein, the food allergy comprises allergy to soy, wheat, eggs, dairy,peanuts, tree nuts, shellfish, fish, mushrooms, stone fruits and otherfruits.

In another embodiment of this aspect and all other aspects providedherein, the composition is administered before the first exposure to apotential food allergen.

In another embodiment of this aspect and all other aspects providedherein, the composition is administered upon clinical signs of atopicsymptoms.

In another embodiment of this aspect and all other aspects providedherein, the composition is administered to individuals with diagnosedfood allergy

In another embodiment of this aspect and all other aspects providedherein, the subject is pretreated with an antibiotic.

In another embodiment of this aspect and all other aspects providedherein, the subject is not pretreated with an antibiotic.

Another aspect described herein relates to a method for reducing oreliminating a subject's immune reaction to a food antigen, the methodcomprising: administering to a subject a composition as describedherein, thereby reducing or eliminating a subject's immune reaction to afood allergen.

In one embodiment of this aspect and all other aspects provided herein,the strains of bacteria administered are selected from the groupconsisting of: Clostridium ramosum, Clostridium scindens, Clostridiumhiranonsis, Clostridium bifermentans, Clostridium leptum, Clostridiumsardiniensis, Bacteroides fragilis, Bacteroides thetaiotaomicron,Bacteroides ovatus, Parabacteroides goldsteinii, Prevotella tannerae,Clostridum hathewayi, Clostridum nexile, Clostridium hylemonae,Clostridium glycyrrhizinilyticum, Clostridium scindens, Clostridiumlavalense, Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable gut bacteria are selected from the groupconsisting of: Clostridium ramosum, C. scindens, C. hiranonsis, C.bifermentans, C. leptum, C. sardiniensis, Bacteroides fragilis, B.thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii, and Prevotellatannerae.

In another embodiment of this aspect and all other aspects providedherein, the strains of viable gut bacteria are selected from the groupconsisting of: (i) Clostridium ramosum, C. scindens, C. hiranonsis, C.bifermentans, C. leptum, and C. sardiniensis, or (ii) Bacteroidesfragilis, B. thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii,and Prevotella tannerae.

In another embodiment of this aspect and all other aspects providedherein, the composition is administered by oral administration, enema,suppository, or orogastric tube.

In another embodiment of this aspect and all other aspects providedherein, the minimal microbial consortium or the viable, culturable,anaerobic gut bacterial strain(s) is/are isolated and/or purified from asubject known to be tolerant to a selected food allergen.

In another embodiment of this aspect and all other aspects providedherein, the strains of bacteria or minimal microbial consortium iscultured and/or maintained under anaerobic conditions.

In another embodiment of this aspect and all other aspects providedherein, anaerobic conditions comprise one or more of the following: (i)oxygen impermeable capsules, (ii) addition of N-acetylcysteine,cysteine, methylene blue or a reducing factor to the composition, or(iii) use of spores for organisms that sporulate.

In another embodiment of this aspect and all other aspects providedherein, the composition administered further comprises a pre-bioticcomposition.

In another embodiment of this aspect and all other aspects providedherein, the composition is enteric-coated.

In another embodiment of this aspect and all other aspects providedherein, the treatment prevents and/or reverses TH2 programming of Tregs.

In another embodiment of this aspect and all other aspects providedherein, the subject is a human subject.

In another embodiment of this aspect and all other aspects providedherein, the method further comprises a step of diagnosing the subject ashaving an IgE-mediated food allergy.

In another embodiment of this aspect and all other aspects providedherein, the method further comprises a step of testing a fecal samplefrom the subject for the presence and/or levels of the bacteria in theminimal microbial consortium or viable, culturable, anaerobic gutbacterial strain(s).

In another embodiment of this aspect and all other aspects providedherein, the food allergy comprises allergy to soy, wheat, eggs, dairy,peanuts, tree nuts, shellfish, fish, mushrooms, stone fruits or otherfruits.

In another embodiment of this aspect and all other aspects providedherein, the composition is administered after an initial exposure and/orreaction to a potential food allergen.

In another embodiment of this aspect and all other aspects providedherein, the biomass of each of the microbes in the administeredcompositions is greater than the biomass of each of the microbesrelative to a reference.

In another embodiment of this aspect and all other aspects providedherein, the subject is pretreated with an antibiotic.

In another embodiment of this aspect and all other aspects providedherein, the subject is not pretreated with an antibiotic.

In another embodiment of this aspect and all other aspects providedherein, the subject is pretreated with a fasting period not longer than24 hours.

Another aspect provided herein relates to a method of monitoring asubject's microbiome, the method comprising: determining the presenceand/or biomass of at least one member of a minimal microbial consortiumin a biological sample obtained from a subject, and wherein if the atleast one member is absent or the biomass of the at least one member islow relative to a reference, the subject is treated with the compositionas described herein.

In another embodiment of this aspect and all other aspects providedherein, the method further comprises predicting that a subject will havean immune response to a food allergen when the at least one member isabsent, the biomass of the at least one member is low relative to areference, or at least one member of a dysbiotic species is present, oris elevated relative to a reference.

In another embodiment of this aspect and all other aspects providedherein, the method is repeated at least one additional time.

In another embodiment of this aspect and all other aspects providedherein, the biological sample is a fecal sample.

Another aspect described herein relates to a synergistic microbialcomposition comprising: (a) a first microbial consortium consistingessentially of four to six strains of viable, non-pathogenic gutbacteria, wherein the strains of viable non-pathogenic gut bacteria areselected from the group consisting of: Clostridium ramosum, C. scindens,C. hiranonsis, C. bifermentans, C. leptum, and C. sardiniensis, and (b)a second microbial consortium consisting essentially of four to fivestrains of viable, non-pathogenic gut bacteria, wherein the strains ofviable non-pathogenic gut bacteria are selected from the groupconsisting of: Bacteroides fragilis, B. thetaiotaomicron, B. ovatus,Parabacteroides goldsteinii, and Prevotella tannerae, wherein one ormore members of the second microbial consortium increases thecolonization and/or persistence of one or more members of the firstmicrobial consortium in a mammalian host.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Tolerance failure in food allergy. Food allergy is a failure oforal tolerance to food antigens associated with Th2 immunity andallergen-specific IgE responses.

FIG. 2. Experimental model: Il4raF709 mutant mice.

FIG. 3. An exemplary ovalbumin sensitization protocol.

FIG. 4. Ovalbumin (OVA)-induced food allergic reaction in Il4raF709mice.

FIG. 5. Allergen-specific TR cell deficiency in allergic Il4raF709 mice.

FIG. 6. Oral allergic sensitization in F709 mutant mice is associatedwith dysbiosis.

FIG. 7. An exemplary protocol to test if microbiota of sensitizedIl4raF709 mice transmit susceptibility to food allergy.

FIG. 8. The microbiota of Il4raF709 mice promotes allergic sensitizationand anaphylaxis in germ free mice.

FIG. 9. The microbiota of Il4raF709 mice promotes allergic sensitizationand anaphylaxis.

FIG. 10. An exemplary protocol to determine if microbiota of foodtolerant mice transmits protection against food allergy.

FIG. 11A-FIG. 11D. The microbiota of food tolerant mice protect againstallergic sensitization and anaphylaxis in a genetically susceptiblehost.

FIG. 12A-FIG. 12D. The microbiota of food tolerant mice promotes theformation of allergen-specific Treg cells.

FIG. 13. A graphical visualization of relative abundances of phyla: 8weeks ovalbumin (OVA) treatment.

FIG. 14. Selected OTUs showing differences between WT and F709 mice:OVA, duodenum, jejunum, ileum.

FIG. 15. An exemplary protocol for testing whether treatment withdefined bacterial mixes will protect against food allergy.

FIG. 16A-FIG. 16D. Clostridia and Bacteriodetes protect againstdevelopment of allergen-specific responses and anaphylaxis.

FIG. 17. Oral allergic sensitization is associated with TR cell TH2reprogramming.

FIG. 18A-FIG. 18F. Deletion of Il4/Il13 in TR cells protects againstfood allergy.

FIG. 19. Reduced TH2-skewed Treg phenotype indicating that Clostridiaand Bacteriodetes have two different molecular mechanisms of action.

FIG. 20A-FIG. 20C. Short chain fatty acid (SCFA) therapy does not rescuefood allergy in Il4raF709 mice.

FIG. 21A-FIG. 21I. Gut protect (GP)-I and GP-II are effective intreat-to-prevent food allergy in conventional wild-type and IL4RA F709mice. FIG. 21A, Conventional IL4raF709 mice are pre-treated with oralbroad-spectrum antibiotics 1 week prior to initiating OVA sensitization.Mice receive weekly doses of 5×10⁸ CFU of the aggregate consortia ofeither GP-1, GP-II or NCC before oral OVA challenge. Mice receive thefinal OVA challenge at 8 weeks, after which temperature drop (FIG. 21B),from anaphylaxis, impact to IgE titers (FIG. 21C), mast cell protease-1(FIG. 21D), mast cell recruitment to the small bowel (FIG. 21E),development of Foxp3+ regulatory T cells (FIG. 21F), and total numbers(FIG. 21G), and interferon gamma vs. IL-4 producing T cells (FIGS. 21H &21I), are measured.

FIG. 22A-FIG. 22H. GP-1 (Clostridiales) and GP-II (Bacteroidetes)consortia protect germ-free mice in treat-to-prevent regimens.Gnotobiotic mice inoculated with either the GP-I or GP-II consortia (onetime) prior to OVA sensitization (left arrow, FIG. 22A) and finalchallenge (right arrow, FIG. 22A).

FIG. 23A-FIG. 23H. GP-I and GP-II consortia cure food allergy inconventional IL4raF709 mice while negative control consortia (NCC) doesnot. FIG. 23A, Conventional IL4raF709 mice are sensitized to OVA for 8weeks and then given oral antibiotics prior to challenge over 4 weekswith the GP-I (Clostridiales), GP-II (Bacteroidetes) or NCC(Proteobacteria) consortia. Afterward, mice receive a final OVAchallenge and the following analyses: FIG. 23B, temperature changesafter OVA challenge as a clinical marker of anaphylaxis. FIG. 23C,Changes to total and OVA-specific IgE titers. FIG. 23D, MMCP-1production (indicative of mast cell degranulation). FIGS. 23E & 23F,Effect of consortia on development of mucosal Foxp3+ CD4+ T cells. FIGS.23G & 23H, Effects on IL-4 and interferon gamma (IFNγ) production inmucosal CD4+ T cells.

FIG. 24A-FIG. 24L. GP-II consortia (Bacteriodetes) protects against foodallergy without prior antibiotic knockdown of the flora.

FIG. 25A-FIG. 25I. GP-I consortia (Clostridiales) can cure food allergywithout the use of antibiotics in conventional mice. FIG. 25A,Conventional wild-type or IL4raF709 mice were challenged weekly with OVAfor 8 weeks, after which they received 5×10⁸ CFU of the GP-I consortium8 times over 4 weeks prior to final OVA challenge. Mice received noprior antibiotic knockdown of the underlying microbiota prior to GP-Iadministration. FIG. 25B, Temperature changes with final OVA challenge.FIG. 25C, Changes in total and OVA-specific IgE titers. FIG. 25D,Changes in percentages of mucosal T cells, Foxp3 regulatory T cells.FIG. 25E, IL4+ mucosal cells and regulatory T cells. FIGS. 25F & 25G,Changes in CD4+ IL-17 production in wild-type and IL4raF709 micereceiving PBS or GP-I consortia. FIGS. 25H & 25I, Changes in IL-17production of Foxp3+ regulatory cells.

DETAILED DESCRIPTION Definitions

As used herein, the term “food allergy” refers to a failure of oraltolerance to food antigens associated with Th2 immunity andallergen-specific IgE responses. That is, an immune response isgenerated in response to particular food antigens and can lead to hives,gastrointestinal symptoms, abdominal pain, anaphylaxis and even death.

As used herein, the term “microbiota” can refer to the human microbiome,the human microbiota, or the human gut microbiota. The human microbiome(or human microbiota) may be understood as the aggregate ofmicroorganisms that reside on the surface and in deep layers of skin, inthe saliva and oral mucosa, in the conjunctiva, and in the genitourinaryand gastrointestinal tracts of humans. The human microbiome is comprisedof bacteria, fungi, viruses, and archaea. At least some of theseorganisms perform tasks that are useful for the human host. Under normalcircumstances, these microorganisms do not cause acute disease to thehuman host, but instead cause no harm or participate in maintaininghealth. Hence, this population of organisms is frequently referred to asthe “normal flora.” The population of microorganisms living in the humangastrointestinal tract is commonly referred to as “microbial flora”,“gut flora”, and/or “gut microbiota”. The microbial flora of the humangut encompasses a wide variety of microorganisms that aid in digestion,the synthesis of vitamins and other metabolites, and creating enzymesnot produced by the human body.

As used herein, the term “minimal microbial consortium” refers to amixed population of cells comprising at least two species of gutbacteria that do not promote acute disease in a subject. The microbialconsortium is “minimal” when an additional bacterial species is addedand there is no additional benefit (e.g., less than 5%) in avoiding ormitigating an allergic response. In some embodiments, the minimalmicrobial consortium comprises at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,or more different species of bacteria. In some embodiments, the minimalmicrobial consortium comprises at least one species of bacteria from thephyla Clostridia and/or Bacteroidetes.

“Operational taxonomic unit (OTU, plural OTUs)” refers to a terminalleaf in a phylogenetic tree and is defined by a specific geneticsequence and all sequences that share a specified degree of sequenceidentity to this sequence at the level of species. A “type” or aplurality of “types” of bacteria includes an OTU or a plurality ofdifferent OTUs, and also encompasses a strain, species, genus, family ororder of bacteria. The specific genetic sequence may be the 16S rRNAsequence or a portion of the 16S rRNA sequence, or it may be afunctionally conserved housekeeping gene found broadly across theeubacterial kingdom. OTUs generally share at least 95%, 96%, 97%, 98%,or 99% sequence identity. OTUs are frequently defined by comparingsequences between organisms. Sequences with less than the specifiedsequence identity (e.g., less than 97%) are not considered to form partof the same OTU.

“Clade” refers to the set of OTUs or members of a phylogenetic treedownstream of a statistically valid node in a phylogenetic tree. Theclade comprises a set of terminal leaves in the phylogenetic tree thatis a distinct monophyletic evolutionary unit.

In microbiology, “16S sequencing” or “16S rRNA” or “16S-rRNA” or “16S”refers to sequence derived by characterizing the nucleotides thatcomprise the 16S ribosomal RNA gene(s). The bacterial 16S rDNA isapproximately 1500 nucleotides in length and is used in reconstructingthe evolutionary relationships and sequence similarity of one bacterialisolate to a second isolate using phylogenetic approaches. 16S sequencesare used for phylogenetic reconstruction as they are in general highlyconserved, but contain specific hypervariable regions that harborsufficient nucleotide diversity to differentiate genera and species ofmost bacteria, as well as fungi.

The “V1-V9 regions” of the 16S rRNA refers to the first through ninthhypervariable regions of the 16S rRNA gene that are used for genetictyping of bacterial samples. These regions in bacteria are defined bynucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043,1117-1173, 1243-1294 and 1435-1465 respectively using numbering based onthe E. coli system of nomenclature. Brosius et al., Complete nucleotidesequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS75(10):4801-4805 (1978). In some embodiments, at least one of the V1,V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize anOTU. In one embodiment, the V1, V2, and V3 regions are used tocharacterize an OTU. In another embodiment, the V3, V4, and V5 regionsare used to characterize an OTU. In another embodiment, the V4 region isused to characterize an OTU. A person of ordinary skill in the art canidentify the specific hypervariable regions of a candidate 16S rRNA bycomparing the candidate sequence in question to the reference sequenceand identifying the hypervariable regions based on similarity to thereference hypervariable regions.

“Dysbiosis” refers to a state of the microbiota or microbiome of the gutor other body area, including mucosal or skin surfaces in which thenormal diversity and/or function of the ecological network is disrupted.Any disruption from the preferred (e.g., ideal) state of the microbiotacan be considered a dysbiosis, even if such dysbiosis does not result ina detectable decrease in health. This state of dysbiosis may beunhealthy, it may be unhealthy under only certain conditions, or it mayprevent a subject from becoming healthier. Dysbiosis may be due to adecrease in diversity, the overgrowth of one or more pathogens orpathobionts, symbiotic organisms able to cause disease only when certaingenetic and/or environmental conditions are present in a patient, or theshift to an ecological network that no longer provides a beneficialfunction to the host and therefore no longer promotes health.

The terms “patient”, “subject” and “individual” are used interchangeablyherein, and refer to an animal, particularly a human, to whom treatment,including prophylactic treatment is provided. The term “subject” as usedherein refers to human and non-human animals. The term “non-humananimals” and “non-human mammals” are used interchangeably hereinincludes all vertebrates, e.g., mammals, such as non-human primates,(particularly higher primates), sheep, dog, rodent (e.g. mouse or rat),guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such aschickens, amphibians, reptiles etc. In one embodiment, the subject ishuman. In another embodiment, the subject is an experimental animal oranimal substitute as a disease model. In another embodiment, the subjectis a domesticated animal including companion animals (e.g., dogs, cats,rats, guinea pigs, hamsters etc.).

As used herein, the term “enteric coated drug delivery device” or“enteric coated composition” refers to any drug delivery method that canbe administered orally but is not degraded or activated until the deviceenters the intestines. Such methods can utilize a coating orencapsulation that is degraded using e.g., pH dependent means,permitting protection of the delivery device and the microbialconsortium to be administered or transplanted throughout the uppergastrointestinal tract until the device reaches the alkaline pH of theintestines. In one embodiment, the enteric coated drug delivery devicecomprises a capsule or a pill. Such drug delivery devices are known tothose of skill in the art.

As used herein, a “prebiotic” refers to an ingredient that allows orpromotes specific changes, both in the composition and/or activity inthe gastrointestinal microbiota that may (or may not) confer benefitsupon the host. In some embodiments, a prebiotic can include one or moreof the following: fructooligosaccharide, galactooligosaccharides,hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan),inulin, chitin, lactulose, mannan oligosaccharides,oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic andcarrageenan), oligofructose, oligodextrose, tagatose, resistantmaltodextrins (e.g., resistant starch), trans-galactooligosaccharide,pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, andrhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugarbeet fiber,pea fiber, corn bran, and oat fiber) and xylooligosaccharides.

As used herein, the terms “administering,” “introducing” and“transplanting” are used interchangeably in the context of the placementof cells, e.g. a microbial consortium, as described herein into asubject, by a method or route which results in at least partiallocalization of the introduced cells at a desired site, such as theintestines or a region thereof, such that a desired effect(s) isproduced (e.g., tolerance to a food allergen). The cells can beadministered by any appropriate route which results in delivery to adesired location in the subject where at least a portion of thedelivered cells or components of the cells remain viable. The period ofviability of the cells after administration to a subject can be as shortas a few hours, e.g., twenty-four hours, to a few days, to as long asseveral years, i.e., long-term engraftment.

As used herein “preventing” or “prevention” refers to any methodologywhere the disease state does not occur due to the actions of themethodology (such as, for example, administration of a compositioncomprising a microbial consortium as described herein). In one aspect,it is understood that prevention can also mean that the disease is notestablished to the extent that occurs in untreated controls. Forexample, there can be a 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80,90, or 100% reduction in the establishment of disease frequency relativeto untreated controls. Accordingly, prevention of a disease encompassesa reduction in the likelihood that a subject will develop the disease,relative to an untreated subject (e.g. a subject who is not treated witha composition comprising a microbial consortium as described herein).

As used herein, the term “full complement of bile acid transformations”refers to the metabolism of primary bile acids to secondary bile acids.Bile acid transformations performed by gut microbes includedeconjugation, deglucuronidation, oxidation of hydroxyl groups,reduction of oxo groups to yield epimeric hydroxyl bile acids,esterification and dehydroxylation. These reactions on bile acids arethe full complement of bile acid transformations as the term is usedherein.

“Synergy” or “synergistic interactions” refers to the interaction orcooperation of two or more microbes to produce a combined effect greaterthan the sum of their separate effects. For example, in one embodiment,“synergy” between two or more microbes can result from a first microbesecreting a waste product or metabolite that the second microbe uses tofuel growth or other processes.

As used herein, the term “persistence” refers to the maintenance of oneor more members of the microbial consortium in the gastrointestinaltract at a number, biomass or activity that is at or above the thresholdfor treating and/or preventing food allergy. Persistence can be measuredby obtaining a stool sample to determine the number, biomass, and/oractivity of one or more members of the microbial consortium. In someembodiments, persistence can be measured by obtaining a ratio of themeasured biomass of at least two members of the microbial consortium inthe stool sample.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease or lessening of a property, level, or otherparameter by a statistically significant amount. In some embodiments,“reduce,” “reduction” or “decrease” or “inhibit” typically means adecrease by at least 10% as compared to a reference level (e.g., theabsence of a given treatment) and can include, for example, a decreaseby at least about 10%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or more. As used herein, “reduction” or“inhibition” does not encompass a complete inhibition or reduction ascompared to a reference level. “Complete inhibition” is a 100%inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase of a property, level, or otherparameter by a statically significant amount; for the avoidance of anydoubt, the terms “increased”, “increase” or “enhance” or “activate”means an increase of at least 10% as compared to a reference level, forexample an increase of at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, at least about a 20-fold increase,at least about a 50-fold increase, at least about a 100-fold increase,at least about a 1000-fold increase or more as compared to a referencelevel.

The term “pharmaceutically acceptable” can refer to compounds andcompositions which can be administered to a subject (e.g., a mammal or ahuman) without undue toxicity.

As used herein, the term “pharmaceutically acceptable carrier” caninclude any material or substance that, when combined with an activeingredient, allows the ingredient to retain biological activity and isnon-reactive with the subject's immune system. Examples include, but arenot limited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution, emulsions such as oil/wateremulsion, and various types of wetting agents. The term“pharmaceutically acceptable carriers” excludes tissue culture media.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodologies, protocols, and reagents, etc., describedherein and as such can vary therefrom. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims.

Microbial Flora

Each individual has a personalized gut microbiota including an estimated500 to 5000 or more species of bacteria, fungi, viruses, archaea andother microorganisms, up to 100 trillion individual organisms, thatreside in the digestive tract, providing a host of useful symbioticfunctions, for example, including aiding in digestion, providingnutrition for the colon, producing vitamins, regulating the immunesystem, assisting in defense against exogenous bacteria, modulatingenergy metabolism, and the production of short chain fatty acids(SCFAs), e.g., via dietary carbohydrates, including resistant starchesand dietary fiber, which are substrates for fermentation that produceSCFAs, primarily acetate, propionate, succinate, butyrate, 1,2propanediol or 1,3 propanediol as end products.

An imbalance in the microbial flora found in and on the human body isknown to be associated with a variety of disease states. For example,obesity in both humans and experimental mouse models is associated withalterations in the intestinal microbiota that appear to be pathogenic.In settings of ‘dysbiosis’ or disrupted symbiosis, microbiota functionsthat can be lost or deranged, resulting in increased susceptibility topathogens, include altered metabolic profiles, or induction ofproinflammatory signals that can result in local or systemicinflammation or autoimmunity. In addition, in asthmatic subjects, boththe bacterial burden and bacterial diversity were significantly higheras compared to control subjects, which were also correlated withbronchial hyper-responsiveness. Thus, the intestinal microbiota plays asignificant role in the pathogenesis of many diseases and disorders,including a variety of pathogenic infections of the gut. For instance,patients become more susceptible to pathogenic infections when thenormal intestinal microbiota has been disturbed due to use ofbroad-spectrum antibiotics. Many of these diseases and disorders arechronic conditions that significantly decrease a patient's quality oflife and can be ultimately fatal.

Microbial Consortia

In some embodiments, a microbial consortium comprises at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12 or more different viable, bacterial species, e.g.,15 or more, 20 or more, 25 or more, 30 or more, or even 40 or more. Inone embodiment, a microbial consortium comprises fewer than 40 species,e.g., 30 or fewer species, 25 or fewer species, 20 or fewer species, or15 or fewer species. In another embodiment, a minimal microbialconsortium comprises 12 or less, 11 or less, 10 or less, 9 or less, 8 orless, 7 or less, 6 or less, 5 or less, or 4 or less different viablebacterial species. In one embodiment, at least one (e.g., at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, 8or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2or fewer) of the bacterial constituents is a non-pathogenic bacterialstrain. Also contemplated are consortia of 4 to 40 species, 4 to 30species, 4 to 25 species, 4 to 20 species, 4 to 15 species, 4 to 11species, 5 to 40 species, 5 to 30 species, 5 to 25 species, 5 to 20species, 5 to 15 species, 5 to 11 species, 6 to 40 species, 6 to 30species, 6 to 25 species, 6 to 20 species, 6 to 15 species, 6 to 11species, 7 to 40 species, 7 to 30 species, 7 to 25 species, 7 to 20species, 7 to 15 species, 7 to 11 species, 8 to 40 species, 8 to 30species, 8 to 25 species, 8 to 20 species, 8 to 15 species, 8 to 11species, 9 to 40 species, 9 to 30 species, 9 to 25 species, 9 to 20species, 9 to 15 species, 9 to 11 species, 10 to 40 species, 10 to 30species, 10 to 25 species, 10 to 20 species, 10 to 15 species, or 10 to11 species.

In one embodiment, the at least 1 bacterial constituent of a microbialconsortium is a bacterial strain(s) of viable gut bacteria selected fromthe group consisting of: Clostridium ramosum, Clostridium scindens,Clostridium hiranonsis, Clostridium bifermentans, Clostridium leptum,Clostridium sardiniensis, Bacteroides fragilis, Bacteroidesthetaiotaomicron, Bacteroides ovatus, Parabacteroides goldsteinii,Prevotella tannerae, Clostridum hathewayi, Clostridum nexile,Clostridium hylemonae, Clostridium glycyrrhizinilyticum, Clostridiumscindens, Clostridium lavalense, Clostridum limetarium, Clostridiumsymbiosum, Clostridium sporosphaeroides, Dialister proprionicfaceins,Dialister succinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti.

In another embodiment, the strains of viable gut bacteria are selectedfrom the group consisting of: Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum, C. sardiniensis, Bacteroidesfragilis, B. thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii,and Prevotella tannerae.

In another embodiment, the strains of viable gut bacteria are selectedfrom the group consisting of: (i) Clostridium ramosum, C. scindens, C.hiranonsis, C. bifermentans, C. leptum, and C. sardiniensis, or (ii)Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, Parabacteroidesgoldsteinii, and Prevotella tannerae.

In one embodiment, the strains of viable gut bacteria do not includeEscherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobactercloacae, Bilophila wadsworthia, Alistipes onderdonkii, Desulfovibriospecies, Lactobacillus johnsoni, and Parasutterella excrementihominis.

In another embodiment, the consortium does not comprise bacteria of theGenera Bilophila, Enterobacter, Escherichia, Klebsiella, Proteus,Alistipes, Desulfovibrio, Blautia, or Parasutterella.

In another embodiment, the consortium does not comprise bacteria of theFamilies Desulfovibrionaceae, Enterobacteriaceae, Rikenellaceae, andSutterellaceae.

In another embodiment, the consortium does not comprise bacteria of theFamilies Lactobacillaceae or Enterbacteriaceae.

In another embodiment, the consortium does not comprise bacteria of theOrder Burkholdales, Desulfovibrionates, or Enterobacteriales.

Metabolic Features:

Various features of gut microbes are beneficial for protection from ortherapy for allergy, including food allergy. In the following, featuresand corresponding functions contemplated to render particular species ortaxa of gut microbes well-suited for a protective or therapeuticmicrobial consortium as described herein are described. In practice, aconsortium comprising four or more, e.g., five or more, six or more,seven or more, eight or more, nine or more or ten or more of thesefeatures and corresponding functions is considered a likely candidatefor protection or therapy for food allergy.

In some embodiments, the microbial consortium comprises one or moretypes of microbes capable of producing butyrate in a mammalian subject.Butyrate-producing microbes can be identified experimentally, e.g., byNMR or gas chromatography analyses of microbial products or colorimetricassays (Rose I A. 1955. Methods Enzymol. 1: 591-5). Butyrate-producingmicrobes can also be identified computationally, e.g., by theidentification of one or more enzymes involved in butyrate synthesis.Non-limiting examples of enzymes found in butyrate-producing microbesinclude butyrate kinase, phosphotransbutyrylase, and butyryl CoA:acetateCoA transferase (Louis P., et al. 2004. J Bact. 186(7): 2099-2106).Butyrate-producing strains include, but are not limited to, Clostridiumsardiniensis, Clostridium hiranonsis, Facealibacterium prausnitzii,Butyrovibrio spp., Eubacterium rectale, and Roseburia intestinalis.

In some embodiments, a pharmaceutical composition comprises two or moretypes of microbes or bacterial strains, wherein the at least two typesof microbes are capable of producing butyrate in a mammalian subject. Inother embodiments, the composition comprises two or more types ofmicrobes, wherein two or more types of microbe cooperate (i.e.,cross-feed) to produce an immunomodulatory short chain fatty acid (SCFA)(e.g., butyrate) in a mammalian subject. In one embodiment, thecomposition comprises at least one type of microbe (e.g.,Bifidobacterium spp., Bacteroides thetaiotaomicron, Bacteroides fragilisor Clostridium ramosum) is capable of metabolizing a prebiotic,including but not limited to, inulin, inulin-type fructans,fucose-containing glycoconjugates including the H1, H2, Lewis A, B, X,or Y antigens, or oligofructose, such that the resulting metabolicproduct can be converted by a second type of microbe (e.g., abutyrate-producing microbe such as Roseburia spp.) to animmunomodulatory SCFA such as butyrate (Falony G., E T al. 2006 Appl.Environ. Microbiol. 72(12): 7835-7841). In other aspects, thecomposition comprises at least one acetate-consuming, butyrate-producingmicrobe (e.g., Faecalibacterium prausnitzii or Roseburia intestinalis).

In some embodiments, the composition comprises one or more types ofmicrobe capable of producing propionate and/or succinate in a mammaliansubject, optionally further comprising a prebiotic or substrateappropriate for propionate and/or succinate biosynthesis. Examples ofprebiotics or substrates used for the production of propionate include,but are not limited to, L-rhamnose, D-tagalose, resistant starch,inulin, polydextrose, arabinoxylans, arabinoxylan oligosaccharides,mannooligosaccharides, and laminarans (Hosseini E., et al. 2011.Nutrition Reviews. 69(5): 245-258). Propionate-producing microbes can beidentified experimentally, such as by NMR or gas chromatography analysesof microbial products or colorimetric assays (Rose I A. 1955. MethodsEnzymol. 1: 591-5). Propionate-producing microbes can also be identifiedcomputationally, such as by the identification of one or more enzymesinvolved in propionate synthesis. Non-limiting examples of enzymes foundin propionate-producing microbes include enzymes of the succinatepathway, including but not limited to phosphoenylpyruvate carboxykinase,pyruvate kinase, pyruvate carboxylase, malate dehydrogenase, fumaratehydratase, succinate dehydrogenase, succinyl CoA synthetase,methylmalonyl Coa decarboxylase, and propionate CoA transferase, as wellas enzymes of the acrylate pathway, including but not limited toL-lactate dehydrogenase, propionate CoA transferase, lactoyl CoAdehydratase, acyl CoA dehydrogenase, phosphate acetyltransferase, andpropionate kinase. For example, microbes that utilize the succinatepathway include certain species of the Bacteroides genus, such asBacteroides fragilis, Clostridium sardiniensis and Clostridumhiranonsis. In one embodiment, the propionate-producing species isBacteroides fragilis, Bacteroides thetaiotaomicron, or Bacteroidesovatus. In one embodiment, the succinate-producing species isBacteroides fragilis, Bacteroides thetaiotaomicron, or Bacteroidesovatus.

Functional methods to define species that produce butyrate, propionateand/or succinate includes analysis of short-chain fatty acid (SCFA)production using gas-chromatography/liquid chromatography (GC/LC) toidentify propionate, butyrate, and/or succinate or mass spectroscopybased methods to detect these SCFA, as well as 1,2-propanediol, and1,3-propanediol. Studies can be performed in cultured supernatants fromcolonized gnotobiotic mice and from conventional patients and/or animalsamples.

Additional methods for identifying species that produce butyratecomprise those species expressing butyryl-CoA:acetate CoA transferases(But genes) or butyrate kinases (Buk genes) for production of butyratefrom anaerobic fermentation of sugars. In another embodiment, organismsproducing butyrate (from amino acids such as lysine, glutarate or4-aminobutyrate pathways) express enzymes including e.g., L2Hgdh,2-hydroxyglutarate dehydrogenase; Gct, glutaconate CoA transferase (α, βsubunits); HgCoAd, 2-hydroxy-glutaryl-CoA dehydrogenase (α, β, γsubunits); Gcd, glutaconyl-CoA decarboxylase (α, β subunits); Th1,thiolase; hbd, β-hydroxybutyryl-CoA dehydrogenase; Cro, crotonase; Bcd,butyryl-CoA dehydrogenase (including electron transfer protein α, βsubunits); KamA, lysine-2,3-aminomutase; KamD,E,β-lysine-5,6-aminomutase (α, β subunits); Kdd, 3,5-diaminohexanoatedehydrogenase; Kce, 3-keto-5-aminohexanoate cleavage enzyme; Kal,3-aminobutyryl-CoA ammonia lyase; AbfH, 4-hydroxybutyrate dehydrogenase;AbfD, 4-hydroxybutyryl-CoA dehydratase; Isom, vinylacetyl-CoA3,2-isomerase (same protein as AbfD): 4Hbt,butyryl-CoA:4-hydroxybutyrate CoA transferase; But, butyryl-CoA:acetateCoA transferase; Ato, butyryl-CoA:acetoacetate CoA transferase (α, βsubunits); Ptb, phosphate butyryltransferase; Buk, and butyrate kinase(see e.g., Vital et al. mBIO 5(2):e00889-14).

In some embodiments, a microbial consortium comprises at least onebacterial species that produces compounds capable of stimulating thearyl hydrocarbon (AhR) receptor in gut epithelial cells,antigen-presenting cells and/or T cells. Without wishing to be bound bytheory, stimulation of the AhR receptor can aid in the development ofregulatory T cell processes that can prevent and/or treat food allergy.Some non-limiting examples of compounds that stimulate host arylhydrocarbon receptor pathways include (i) indole, (ii) intermediatesfrom microbial synthesis of indole, tryptophan, tyrosine and histidine,(iii) microbial synthesis of flavonoids, phenazines and/or quinones or(iv) compounds or intermediates of metabolism of host ingestedflavonoids, phenazines and/or quinones. In one example, a viable,culturable, anaerobic gut bacterial strain produces aryl hydrocarbonreceptor agonists sufficient to stimulate host aryl hydrocarbon receptorpathways comprises at least one gene associated with the synthesis oftryptophan or the synthesis of quinone molecules. In an additionalexample, a viable, culturable, anaerobic gut bacterial strain thatproduces aryl hydrocarbon receptor agonists sufficient to stimulate hostaryl hydrocarbon receptor pathways by microbial synthesis of flavonoids,phenazines, and/or quinones. Thus microbes that express or encodebiosynthetic enzymes that participate in the synthesis of flavonoids,phenazines and/or quinones are identified as microbes that produce hostaryl hydrocarbon receptor agonists. In one embodiment, the biosyntheticenzymes include the last enzyme in the pathway that catalyzes the finalbiosynthetic reaction producing e.g., flavonoids, phenazine or quinonecompounds.

In some embodiments, a microbial consortium comprises at least onebacterial species that produces compounds capable of stimulating thepregnane X receptor that e.g., has beneficial effects on gut barrierfunction and/or the development of regulatory T cell processes.Non-limiting examples of compounds that stimulate the pregnane Xreceptor include (i) desmolase, (ii) compounds or intermediates ofhydroxysteroid dehydrogenase activity, or (iii) compounds orintermediates derived from flavonoid metabolism enzymes. Thus, bacteriathat encode and express steroid desmolase and/or hydroxysteroiddehydrogenase enzymes are expected to produce compounds that stimulatethe pregnane X receptor. Clostridium sardiniensis and Clostridiumscindens are non-limiting examples of bacterial species that producecompounds capable of stimulating the pregnane X receptor.

In some embodiments, the microbial consortium comprises at least onebacterial species that produces compounds capable of stimulating theRAR-related orphan receptor gamma (RORgamma) pathways, for example, tostimulate development of regulatory T cell responses via directstimulation of RORgamma-activated pathways in gut antigen presentingcells and/or epithelial cells that then stimulate regulatory T cellresponses. In one embodiment, the viable, culturable, anaerobic gutbacterial strain that produces compounds endogenously or by metabolizingingested precursors, that is capable of stimulating the RORgamma(RAR-related orphan receptor gamma) pathways to stimulate development ofregulatory T cell responses is a strain that expresses at least onecholesterol reductase and other enzymes capable of metabolizing sterolcompounds. Non-limiting examples of microbes that produce compounds thatstimulate the RORgamma pathway include Clostridium scindens, Clostridiahiranonsis, and Clostridium sardiniensis. In one instance, those speciesexpress bile acid transforming enzymes that can also produce RORgammapathway agonists.

In some embodiments, a microbial consortium described herein improvesgut function, for example, by stimulating host mucins and complexglycoconjugates and improving colonization by protective commensalstrains. In one embodiment, the microbial consortium comprises at leastone bacterial strain, such as Bacteroides thetaiotaomicron, thatstimulates production of mucins and complex glycoconjugates by the host.

Immunomodulation:

Other exemplary compositions useful for treatment of food allergycontain bacterial strains capable of altering the proportion of immunesubpopulations, e.g., T cell subpopulations, e.g., Tregs in the subject.

For example, immunomodulatory bacteria can increase or decrease theproportion of Treg cells, Th17 cells, Th1 cells, or Th2 cells in asubject. The increase or decrease in the proportion of immune cellsubpopulations can be systemic, or it can be localized to a site ofaction of the colonized consortium, e.g., in the gastrointestinal tractor at the site of a distal dysbiosis. In some embodiments, a microbialconsortium comprising immunomodulatory bacteria is used for treatment offood allergy based on the desired effect of the probiotic composition onthe differentiation and/or expansion of subpopulations of immune cellsin the subject.

In one embodiment, the microbial consortium contains immunomodulatorybacteria that increase the proportion of Treg cells in a subject or in aparticular location in a subject, e.g., the gut tissues. In oneembodiment, a microbial consortium contains immunomodulatory bacteriathat increase the proportion of Th17 cells in a subject. In anotherembodiment, a microbial consortium contains immunomodulatory bacteriathat decrease the proportion of Th17 cells in a subject. In oneembodiment, a microbial consortium contains immunomodulatory bacteriathat increase the proportion of Th1 cells in a subject. In anotherembodiment, a microbial consortium contains immunomodulatory bacteriathat decrease the proportion of Th1 cells in a subject. In oneembodiment, a microbial consortium contains immunomodulatory bacteriathat increase the proportion of Th2 cells in a subject. In anotherembodiment, a microbial consortium contains immunomodulatory bacteriathat decrease the proportion of Th2 cells in a subject.

In one embodiment, a microbial consortium contains immunomodulatorybacteria capable of modulating the proportion of one or more of Tregcells, Th17 cells, Th1 cells, Th2 cells, and combinations thereof in asubject. Certain immune cell profiles can be particularly desirable totreat or prevent inflammatory disorders, such as food allergies. Forexample, in some embodiments, treatment or prevention of e.g., foodallergy can be promoted by increasing numbers of Treg cells and Th2cells, and decreasing numbers of Th17 cells and Th1 cells. Accordingly,a microbial consortium for the treatment or prevention of food allergycan contain a microbial consortium capable of promoting Treg cells andTh2 cells, and reducing Th17 and Th1 cells.

In one embodiment, the anaerobic gut bacterial strain in the methods andcompositions described herein express agonists capable of binding to andmodulating responses mediated by Toll-like receptors (TLR), CD14 and/orlipid binding proteins in antigen presenting cells, gut epithelial cellsand/or T cells to promote the development of regulatory T cells.Non-limiting examples of TLR agonists include lipopolysaccharide (LPS),exopolysaccharides (PSA), peptidoglycan or CpG motifs produced bycommensal members of Bacteroides, or lipoteichoic acids (LTA) producedby members of Clostridium. In one embodiment, an anaerobic gut bacterialstrain that acts as a TLR agonist is selected from the following Table.

Family Genus Species Clostridieaceae Clostridium, Hungatella Hungatellahathawayi Eubacteriaceae Eubacterium Eubacterium rectaleErysipelotrichaceae Erysipelatoclostridium Erysipelatoclostridium(formerly species in ramosum (Clostridium genus Clostridium) ramosum)Lachnospiraceae Blautia, Butyrovibrio, Butyrovibrio crossatus,Cellulosyliticum, Roseburia intestinalis, Clostridium clusterClostridium scindens, XIVa species, Clostridium hylemonae, Coprococcus,Dorea, Clostridium symbiosum Lachnospira, Robinsonella, Roseburia,Ruminococcaceae Faecalobacterium, Faecalibacterium Ruminococcus,prausnitzii, Subdoligranulum, Subdoligranulum Clostridium clustervariabile XIVa species Bacteroidaceae Bacteroides Bacteroidesthetaiotaomicron, Bacteroides fragilis, Bacteroides ovatusProphyromonadaceae Parabacteroides, Parabacteroides Porphyromonas,goldsteinii, Tannerella Parabacteroides merdae, Parabacteroidesdistasonis Prevotellaceae Prevotella Prevotella tannerae

Bile Acid Transformation:

Primary bile acids (e.g., cholic and chenodeoxycholic acids in humans)are generated in the liver of mammals, including humans, mainly byconjugation with the amino acids taurine or glycine, and are secreted inbile. In the intestinal tract, primary bile acids are metabolized bymicrobes that transform the primary bile acids to secondary bile acids.Intestinal microbial transformation of primary bile acids can includedeconjugation, deglucuronidation, oxidation of hydroxyl groups,reduction of oxo groups to yield epimeric hydroxyl bile acids,esterification, and dehydroxylation. Non-limiting examples of bacteriathat perform deconjugation of primary bile acids include Bacteroides,Bifidobacterium, Clostridium, and Lactobacillus. Non-limiting examplesof bacteria that perform oxidation and epimerization of primary bileacids include Bacteroides, Clostridium, Egghertella, Eubacterium,Peptostreptococcus, and Ruminococcus. Non-limiting examples of bacteriathat perform 7-dehydroxylation of primary bile acids includeClostridium, and Eubacterium. Non-limiting examples of bacteria thatperform esterification of primary bile acids include Bacteroides,Eubacterium, and Lactobacillus.

In one embodiment, a microbial consortium as described herein comprisesat least one bacterial constituent that transforms bile acids bydeconjugation. In another embodiment, a microbial consortium asdescribed herein comprises at least one bacterial constituent thattransforms bile acids by 7-dehydroxylation. In another embodiment, amicrobial consortium as described herein comprises at least onebacterial constituent that transforms bile acids by esterification.

In one embodiment, a microbial consortium as described herein comprisesat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11 or more bacterialconstituents that perform bile acid transformation.

In one embodiment, a microbial consortium as described herein comprises11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 orfewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer 1 or fewer or zerobacterial constituents that perform bile acid transformation, such asdeconjugation, esterification or 7-dehydroxylation.

In one embodiment, a microbial consortium comprises at least oneanaerobic gut bacterial strain that alone, or in combination, performsthe full complement of bile acid transformations.

Engineered Microbes:

In some embodiments, one or more members of the microbial consortiumcomprises an engineered microbe(s). For example, engineered microbesinclude microbes harboring i) one or more introduced genetic changes,such change being an insertion, deletion, translocation, orsubstitution, or any combination thereof, of one or more nucleotidescontained on the bacterial chromosome or on an endogenous plasmid,wherein the genetic change can result in the alteration, disruption,removal, or addition of one or more protein coding genes,non-protein-coding genes, gene regulatory regions, or any combinationthereof, and wherein such change can be a fusion of two or more separategenomic regions or can be synthetically derived; ii) one or more foreignplasmids containing a mutant copy of an endogenous gene, such mutationbeing an insertion, deletion, or substitution, or any combinationthereof, of one or more nucleotides; and iii) one or more foreignplasmids containing a mutant or non-mutant exogenous gene or a fusion oftwo or more endogenous, exogenous, or mixed genes. The engineeredmicrobe(s) can be produced using techniques including but not limited tosite-directed mutagenesis, transposon mutagenesis, knock-outs,knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis,ultraviolet light mutagenesis, transformation (chemically or byelectroporation), phage transduction, or any combination thereof.

Excluded Bacteria:

In one embodiment, a microbial consortium does not include an organismconventionally classified as a pathogenic or opportunistic organism. Itis possible that a function shared by all members of a given taxonomicgroup could be beneficial, e.g., for providing particular metabolites,yet for other reasons the overall effect of one or more particularmembers of the group is not beneficial and is, for example, pathogenic.Clearly, members of a given taxonomic group that cause pathogenesis,e.g., acute gastrointestinal pathologies, are to be excluded from thetherapeutic or preventive methods and compositions described herein.

In one embodiment, the bacterial composition does not comprise at leastone of: Acidaminococcus intestinalis, Escherichia coli, Lactobacilluscasei, Lactobacillus paracasei, Raoultella sp., and Streptococcus mitis.

In another embodiment, the bacterial composition does not comprise atleast one of Bamesiella intestinihominis; Lactobacillus reuteri;Enterococcus hirae, Enterococus faecium, or Enterococcus durans;Anaerostipes caccae or Clostridium indolis; Staphylococcus wameri orStaphylococcus pasteuri; and Adlercreutzia equolifaciens.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium botulinum, Clostridium cadaveric, Clostridiumchauvoei, Clostridium clostridioforme, Clostridium cochlearium,Clostridium difficile, Clostridium haemolyticum, Clostridium hastiforme,Clostridium histolyticum, Clostridium indolis, Clostridium irregulare,Clostridium limosum, Clostridium malenominatum, Clostridium novyi,Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium septicum, Clostridium sordellii,Clostridium sphenoides, and Clostridium tetani.

In another embodiment, the bacterial composition does not comprise atleast one of Escherichia coli, and Lactobacillus johnsonii.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium innocuum, Clostridium butyricum, Escherichiacoli, and Blautia producta (previously known as Peptostreptococcusproductus).

In another embodiment, the bacterial composition does not comprise atleast one of Eubacteria, Fusobacteria, Propionibacteria, Escherichiacoli, and Gemmiger.

In another embodiment, the compositions described herein do not comprisepathogenic bacteria such as e.g., Yersinia, Vibrio, Treponema,Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia,Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria,Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus,Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella,Corynebacterium, Chlamydia, Chlamydophila, Campylobacter, Burkholderia,Brucella, Borrelia, Bordetella, Bacillus, multi-drug resistant bacteria,extended spectrum beta-lactam resistant Enterococci (ESBL),Carbapenem-resistant Enterobacteriaceae (CRE), and vancomycin-resistantEnterococci (VRE).

In other embodiments, the compositions described herein do not comprisepathogenic species or strains, such as Aeromonas hydrophila,Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus,Campylobacter jejuni, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, enteroaggregative Escherichia coli,enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli,enterotoxigenic Escherichia coli (such as, but not limited to, LT and/orST), Escherichia coli 0157:H7, Helicobacter pylori, Klebsielliapneumonia, Lysteria monocytogenes, Plesiomonas shigelloides, Salmonellaspp., Salmonella typhi, Salmonella paratyphi, Shigella spp.,Staphylococcus spp., Staphylococcus aureus, vancomycin-resistantenterococcus spp., Vibrio spp., Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica.

In one embodiment, the microbial consortia and compositions thereof donot comprise Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis,Enterobacter cloacae, and/or Bilophila wadsworthia.

Reduction of Pathogenic Organisms:

In some embodiments, compositions comprising a microbial consortium asdescribed herein offer a protective or therapeutic effect againstdysbiosis or against infection by one or more GI pathogens of interest.In one embodiment, a microbial consortium as described herein reducesthe biomass of one or more dysbiotic or pathogenic bacterial strains.

In one embodiment, a microbial consortium as described herein decreasesthe biomass of one or more dysbiotic or pathogenic bacterial strains byat least 10% compared to the biomass of the one or more dysbiotic orpathogenic bacterial strains in the absence of treatment with suchmicrobial consortium. In other embodiments the biomass of one or morepathogenic bacterial strains is decreased by at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or even 100% (i.e., belowdetectable limits of the assay) as compared to the biomass of thedysbiotic or pathogenic bacterial strains in the gut of the subjectprior to treatment with the microbial consortium or compositionsthereof.

In some embodiments, a microbial consortium as described herein altersthe gut environment such that the number, biomass, or activity of one ormore dysbiotic or pathogenic organisms is decreased by at least 10%(e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or even 100% (i.e., below detectable limits of the assay)). As butone example, colonization of Bacteroides reduces the biomass ofdysbiotic species in the Enterobacteriaceae or DesulfonovibriacaeaFamilies.

In some embodiments, the pathogenic bacterium is selected from the groupconsisting of Yersinia, Vibrio, Treponema, Streptococcus,Staphylococcus, Escherichia/Shigella, Salmonella, Rickettsia, Orientia,Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira,Legionella, Klebsiella, Helicobacter, Haemophilus, Francisella,Escherichia, Ehrlichia, Enterococcus, Coxiella, Corynebacterium,Clostridium, Chlamydia, Chlamydophila, Campylobacter, Burkholderia,Brucella, Borrelia, Bordetella, Bifidobacterium, Bacillus, Bilophila,Desulfovibrio, multi-drug resistant bacteria, extended spectrumbeta-lactam resistant Enterococci (ESBL), Carbapenem-resistantEnterobacteriaceae (CRE), and vancomycin-resistant Enterococci (VRE).

In some embodiments, these pathogens include, but are not limited to,Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides,Bacillus cereus, Campylobacter jejuni, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, enteroaggregativeEscherichia coli, entero hemorrhagic Escherichia coli, enteroinvasiveEscherichia coli, enterotoxigenic Escherichia coli (such as, but notlimited to, LT and/or ST), Escherichia coli 0157:H7, Helicobacterpylori, Klebsiellia pneumonia, Lysteria monocytogenes, Plesiomonasshigelloides, Salmonella spp., Salmonella typhi, Salmonella paratyphi,Shigella spp., Staphylococcus spp., Staphylococcus aureus,vancomycin-resistant enterococcus spp., Vibrio spp., Vibrio cholerae,Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica.

In one embodiment, the pathogen of interest is at least one pathogenchosen from Clostridium difficile, Salmonella spp., pathogenicEscherichia coli, vancomycin-resistant Enterococcus spp., and extendedspectrum beta-lactam resistant Enterococci (ESBL).

Methods for testing the efficacy of the compositions comprising amicrobial composition to reduce the number, biomass, or activity of oneor more dysbiotic or pathogenic organisms are discussed in thefollowing. While certain of the methods are described in the followingin terms of assaying reduced number, biomass or activity of C.difficile, one of skill in the art can readily adapt the methods tomeasure the number, biomass or activity of one or more further microbialstrains.

In one embodiment, provided is an In Vitro Assay utilizing competitionbetween the bacterial compositions or subsets thereof and C. difficileor other dysbiotic or pathogenic strain. This test in known in the artand as such is not described in detail herein.

In another embodiment, provided is an In Vitro Assay utilizing 10%(wt/vol) Sterile-Filtered Feces. This assay tests for the protectiveeffect of the bacterial compositions and screens in vitro forcombinations of microbes that inhibit the growth of a given pathogenicor dysbiotic microbe. The assay can operate in automated high-throughputor manual modes. Under either system, human or animal feces can bere-suspended in an anaerobic buffer solution, such as pre-reduced PBS orother suitable buffer, the particulate removed by centrifugation, andfilter sterilized. This 10% sterile-filtered feces material serves asthe base media for the in vitro assay. To test a bacterial composition,an investigator can add it to the sterile-filtered feces material for afirst incubation period and then can inoculate the incubated microbialsolution with a pathogenic or dysbiotic microbe of interest for a secondincubation period. The resulting titer of the pathogenic or dysbioticmicrobe is quantified by any number of methods such as those describedbelow, and the change in the amount of pathogen is compared to standardcontrols including the pathogenic or dysbiotic microbe cultivated in theabsence of the bacterial composition. The assay is conducted using atleast one control. Feces from a healthy subject can be used as apositive control. As a negative control, antibiotic-treated feces orheat-treated feces can be used. Various bacterial compositions can betested in this material and the bacterial compositions optionallycompared to the positive and/or negative controls. The ability toinhibit the growth of a pathogenic or dysbiotic microbe can be measuredby plating the incubated material on selective media and countingcolonies. After competition between the bacterial composition and thepathogenic or dysbiotic microbe, each well of the in vitro assay plateis serially diluted ten-fold six times, and plated on selective media.For Clostridium difficile this would include, for example, cycloserinecefoxitin mannitol agar (CCMA) or cycloserine cefoxitin fructose agar(CCFA), and incubated. Colonies of the pathogenic or dysbiotic microbesare then counted to calculate the concentration of viable cells in eachwell at the end of the competition.

Alternatively, the ability to inhibit the growth of a pathogenic ordysbiotic species can be measured by quantitative PCR (qPCR). Standardtechniques can be followed to generate a standard curve for thepathogenic or dysbiotic strain of interest. Genomic DNA can be extractedfrom samples using commercially-available kits, such as the Mo BioPowersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories,Carlsbad, Calif.), the Mo Bio Powersoil® DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.The qPCR can be conducted using HotMasterMix (5PRIME, Gaithersburg, Md.)and primers specific for the pathogenic or dysbiotic microbe ofinterest, and can be conducted on a MicroAmp® Fast Optical 96-wellReaction Plate with Barcode (0.1 mL) (Life Technologies, Grand Island,N.Y.) and performed on a BioRad C1000™ Thermal Cycler equipped with aCFX96™ Real-Time System (BioRad, Hercules, Calif.), with fluorescentreadings of the FAM and ROX channels. The Cq value for each well on theFAM channel is determined by the CFX Manager™ software version 2.1. Thelog₁₀ (cfu/ml) of each experimental sample is calculated by inputting agiven sample's Cq value into linear regression model generated from thestandard curve comparing the Cq values of the standard curve wells tothe known log₁₀ (cfu/ml) of those samples. The skilled artisan canemploy alternative qPCR modes.

Also provided are In Vivo Assays establishing the protective effect ofbacterial compositions. The assay is described in terms of protectiveeffect against Clostridium difficile, but can be adapted by one of skillin the art for other pathogens or dysbiotic species. Provided is an invivo mouse model to test for the protective effect of the bacterialcompositions against C. difficile. In this model (based on Chen, et al.,Gastroenterology 135(6):1984-1992 (2008)), mice are made susceptible toC. difficile by a 7 day treatment (days −12 to −5 of experiment) with 5to 7 antibiotics (including kanamycin, colistin, gentamycin,metronidazole and vancomycin and optionally including ampicillin andciprofloxacin) delivered via their drinking water, followed by a singledose with Clindamycin on day −3, then challenged three days later on day0 with 10⁴ spores of C. difficile via oral gavage (i.e., oro-gastriclavage). Bacterial compositions can be given either before (prophylactictreatment) or after (therapeutic treatment) C. difficile gavage.Further, bacterial compositions can be given after (optional) vancomycintreatment to assess their ability to prevent recurrence and thussuppress the pathogen in vivo. The outcomes assessed each day from day−1 to day 6 (or beyond, for prevention of recurrence) are weight,clinical signs, mortality and shedding of C. difficile in the feces.Weight loss, clinical signs of disease and C. difficile shedding aretypically observed without treatment. Vancomycin provided by oral gavageon days −1 to 4 protects against these outcomes and serves as a positivecontrol. Clinical signs are subjective, and scored each day by the sameexperienced observer. Animals that lose greater than or equal to 25% oftheir body weight are euthanized and counted as infection-relatedmortalities. Feces are gathered from mouse cages (5 mice per cage) eachday, and the shedding of C. difficile spores is detected in the fecesusing a selective plating assay as described for the in vitro assayabove, or via qPCR for the toxin gene. The effects of test materialsincluding 10% suspension of human feces (as a positive control),bacterial compositions, or PBS (as a negative vehicle control), aredetermined by introducing the test article in a 0.2 mL volume into themice via oral gavage on day −1, one day prior to C. difficile challenge,on day 1, 2 and 3 as treatment or post-vancomycin treatment on days 5,6, 7 and 8. Vancomycin, as discussed above, is given on days 1 to 4 asanother positive control. Alternative dosing schedules and routes ofadministration (e.g. rectal) may be employed, including multiple dosesof test article, and 10³ to 10¹³ of a given organism or composition maybe delivered.

Enhancement of Beneficial Organisms:

In some embodiments, compositions comprising a microbial consortiumoffer a therapeutic effect of enhancing beneficial organisms in the GItract. In one embodiment, a microbial consortium as described hereinincreases the biomass of one or more beneficial bacterial strains by atleast 10%. In other embodiments the biomass of one or more beneficialbacterial strains is increased by at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 1-fold, at least 2-fold, at least 5-fold, at least10-fold, at least 50-fold, at least 100-fold, at least 500-fold, atleast 1000-fold, at least 5000-fold, at least 10,000-fold, at least15,000-fold or at least 20,000-fold over the biomass of the beneficialbacterial strains in the gut of the subject prior to treatment with themicrobial consortium or compositions thereof. In one embodiment, thebeneficial organisms are commensal bacterial strains that currentlyreside or exist in the gut. In another embodiment, the beneficialorganisms are one or more of the bacterial strains in the microbialconsortium itself.

In some embodiments, a microbial consortium as described herein altersthe gut environment such that the number, biomass, or activity of one ormore beneficial organisms is increased by at least 10% (e.g., by atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold,at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold,at least 500-fold, at least 1000-fold, at least 5000-fold, at least10,000-fold, at least 15,000-fold or at least 20,000-fold). For example,the microbial consortium stimulates the host's production of mucins andcomplex glycoconjugates to improve gut barrier function and colonizationof beneficial organisms, additional probiotic compositions, or themicrobial consortium itself. In some embodiments, the microbialcomposition for enhancing the biomass and/or activity of beneficialorganisms comprises e.g., Bacteroides thetaiotaomicron, which enhancescolonization by other Bacteroidetes and Clostridiales. In someembodiments, the microbial consortium influences gut pH, reduction ofoxygen tension, secretion of glycosidases, and improving the reductionpotential of the gut lumen to improve the colonization of beneficialorganisms.

In another embodiment, the beneficial species comprises a Clostridiumspp, such as Clostridium ramosum, Clostridium scindens, Clostridiumhiranonsis, Clostridium bifermentans, Clostridium leptum, Clostridiumsardiniensis, Clostridium hathewayi, Clostridium nexile, Clostridiumhylemonae, Clostridium glycyrrhizinilyticum, Clostridium lavalense,Clostridium fimetarium, Clostridium symbiosum, or Clostridiumsporosphaeroides.

Characterization of Bacteria and Bacterial Consortia

In certain embodiments, methods are provided for testing certaincharacteristics of compositions comprising a microbial consortium. Forexample, the sensitivity of bacterial compositions to certainenvironmental variables is determined, e.g., in order to select forparticular desirable characteristics in a given composition, formulationand/or use. For example, the bacterial constituents of the compositioncan be tested for pH resistance, bile acid resistance, and/or antibioticsensitivity, either individually on a constituent-by-constituent basisor collectively as a bacterial composition comprised of multiplebacterial constituents (collectively referred to in this section as amicrobial consortium).

pH Sensitivity Testing:

If a pharmaceutical composition will be administered other than to thecolon or rectum (i.e., for example, an oral route), optionally testingfor pH resistance enhances the selection of microbes or therapeuticcompositions that will survive at the highest yield possible through thevarying pH environments of the distinct regions of the GI tract orgenitourinary tracts. Understanding how the bacterial compositions reactto the pH of the GI or genitourinary tracts also assists in formulation,so that the number of microbes in a dosage form can be increased ifbeneficial and/or so that the composition can be administered in anenteric-coated capsule or tablet or with a buffering or protectivecomposition.

As the pH of the stomach can drop to a pH of 1 to 2 after a high-proteinmeal for a short time before physiological mechanisms adjust it to a pHof 3 to 4 and often resides at a resting pH of 4 to 5, and as the pH ofthe small intestine can range from a pH of 6 to 7.4, bacterialcompositions can be prepared that survive these varying pH ranges(specifically wherein at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or as much as 100% of the bacteria can survivegut transit times through various pH ranges). This can be tested byexposing the bacterial composition to varying pH ranges for the expectedgut transit times through those pH ranges. Therefore, as a non-limitingexample only, 18-hour cultures of compositions comprising one or morebacterial species or strains can be grown in standard media, such as gutmicrobiota medium (“GMM”, see Goodman et al., PNAS 108(15):6252-6257(2011)) or another animal-products-free medium, with the addition of pHadjusting agents for a pH of 1 to 2 for 30 minutes, a pH of 3 to 4 for 1hour, a pH of 4 to 5 for 1 to 2 hours, and a pH of 6 to 7.4 for 2.5 to 3hours. An alternative method for testing stability to acid is describedin e.g., U.S. Pat. No. 4,839,281. Survival of bacteria can be determinedby culturing the bacteria and counting colonies on appropriate selectiveor non-selective media.

Bile Acid Sensitivity Testing:

Additionally, in some embodiments, testing for bile-acid resistanceenhances the selection of microbes or therapeutic compositions that willsurvive exposures to bile acid during transit through the GI tract. Bileacids are secreted into the small intestine and can, like pH, affect thesurvival of bacterial compositions. This can be tested by exposing thecompositions to bile acids for the expected gut exposure time to bileacids. For example, bile acid solutions can be prepared at desiredconcentrations using 0.05 mM Tris at pH 9 as the solvent. After the bileacid is dissolved, the pH of the solution can be adjusted to 7.2 with10% HCl. Bacterial components of the therapeutic compositions can becultured in 2.2 ml of a bile acid composition mimicking theconcentration and type of bile acids in the patient, 1.0 ml of 10%sterile-filtered feces media and 0.1 ml of an 18-hour culture of thegiven strain of bacteria. Incubations can be conducted for from 2.5 to 3hours or longer. An alternative method for testing stability to bileacid is described in e.g., U.S. Pat. No. 4,839,281. Survival of bacteriacan be determined by culturing the bacteria and counting colonies onappropriate selective or non-selective media.

Antibiotic Sensitivity Testing:

As a further optional sensitivity test, the bacterial components of themicrobial compositions can be tested for sensitivity to antibiotics. Inone embodiment, the bacterial components can be chosen so that they aresensitive to antibiotics such that if necessary they can be eliminatedor substantially reduced from the patient's gastrointestinal tract by atleast one antibiotic targeting the bacterial composition.

Adherence to Gastrointestinal Cells:

The compositions can optionally be tested for the ability to adhere togastrointestinal cells. A method for testing adherence togastrointestinal cells is described in e.g., U.S. Pat. No. 4,839,281.

Identification of Immunomodulatory Bacteria:

In some embodiments, immunomodulatory bacteria are identified by thepresence of nucleic acid sequences that modulate sporulation. Inparticular, signature sporulation genes are highly conserved acrossmembers of distantly related genera including Clostridium and Bacillus.Traditional approaches of forward genetics have identified many, if notall, genes that are essential for sporulation (spo). The developmentalprogram of sporulation is governed in part by the successive action offour compartment-specific sigma factors (appearing in the order σF, σE,σG and σK), whose activities are confined to the forespore (σF and σG)or the mother cell (σE and σK). In other embodiments, immunomodulatorybacteria are identified by the biochemical activity of DPA producingenzymes or by analyzing DPA content of cultures. As part of thebacterial sporulation, large amounts of DPA are produced, and comprise5-15% of the mass of a spore. Because not all viable spores germinateand grow under known media conditions, it is difficult to assess a totalspore count in a population of bacteria. As such, a measurement of DPAcontent highly correlates with spore content and is an appropriatemeasure for characterizing total spore content in a bacterialpopulation.

In other embodiments, immunomodulatory bacteria are identified byscreening bacteria to determine whether the bacteria induce secretion ofpro-inflammatory or anti-inflammatory cytokines by host cells. Forexample, human or mammalian cells capable of cytokine secretion, such asimmune cells (e.g., PBMCs, macrophages, T cells, etc.) can be exposed tocandidate immunomodulatory bacteria, or supernatants obtained fromcultures of candidate immunomodulatory bacteria, and changes in cytokineexpression or secretion can be measured using standard techniques, suchas ELISA, immunoblot, Luminex™, antibody array, quantitative PCR,microarray, etc. Bacteria can be selected for inclusion in a microbialconsortium based on the ability to induce a desired cytokine profile inhuman or mammalian cells. For example, anti-inflammatory bacteria can beselected for inclusion (or alternatively exclusion) in a microbialconsortium or composition thereof, based on the ability to inducesecretion of one or more anti-inflammatory cytokines, and/or the abilityto reduce secretion of one or more pro-inflammatory cytokines.Anti-inflammatory cytokines include, for example, IL-10, IL-13, IL-9,IL-4, IL-5, and combinations thereof. Other inflammatory cytokinesinclude, for example, TGFβ. Pro-inflammatory cytokines include, forexample, IFNγ, IL-12p70, IL-1α, IL-6, IL-8, MCP1, MIP1α, MIP1β, TNFα,and combinations thereof. In some embodiments, anti-inflammatorybacteria can be selected for inclusion in a microbial consortium basedon the ability to modulate secretion of one or more anti-inflammatorycytokines and/or the ability to reduce secretion of one or morepro-inflammatory cytokines by a host cell induced by a bacteria of adifferent type (e.g., a bacteria from a different species or from adifferent strain of the same species).

In other embodiments, immunomodulatory bacteria are identified byscreening bacteria to determine whether the bacteria impact thedifferentiation and/or expansion of particular subpopulations of immunecells. For example, candidate bacteria can be screened for the abilityto promote differentiation and/or expansion of Treg cells, Th17 cells,Th1 cells and/or Th2 cells from precursor cells, e.g. naive T cells. Byway of example, naïve T cells can be cultured in the presence ofcandidate bacteria or supernatants obtained from cultures of candidatebacteria, and numbers of Treg cells, Th17 cells, Th1 cells and/or Th2cells can be determined using standard techniques, such as FACSanalysis. Markers indicative of Treg cells include, for example,CD25⁺CD127^(lo). Markers indicative of Th17 cells include, for example,CXCR3 ⁻ CCR6⁺. Markers indicative of Th1 cells include, for example,CD4⁺, CXCR3⁺, and CCR6⁻. Markers indicative of Th2 cells include, forexample, CD4⁺, CCR4⁺, and CXCR3⁻, CCR6⁻. Other markers indicative ofparticular T cells subpopulations are known in the art, and may be usedin the assays described herein, e.g., to identify populations of immunecells impacted by candidate immunomodulatory bacteria. Bacteria can beselected for inclusion (or exclusion) in a microbial consortium based onthe ability to promote differentiation and/or expansion of a desiredimmune cell subpopulation.

In other embodiments, immunomodulatory bacteria are identified byscreening bacteria to determine whether the bacteria secrete short chainfatty acids (SCFA), such as, for example, butyrate, acetate, propionate,or valerate, or combinations thereof. For example, secretion of shortchain fatty acids into bacterial supernatants can be measured usingstandard techniques. In one embodiment, bacterial supernatants can bescreened to measure the level of one or more short chain fatty acidsusing NMR, mass spectrometry (e.g., GC-MS, tandem mass spectrometry,matrix-assisted laser desorption/ionization, etc.), ELISA, orimmunoblot. Expression of bacterial genes responsible for production ofshort chain fatty acids can also be determined by standard techniques,such as Northern blot, microarray, or quantitative PCR.

Exemplary Minimal Microbial Consortia:

Minimal microbial consortia are shown herein in the Examples section andcan prevent and/or treat existing symptoms of a food allergy. Theseexemplary minimal microbial consortia should not be construed aslimiting and are intended only for the better understanding of themethods and compositions described herein.

In one embodiment, a minimal microbial consortium consists essentiallyof: Clostridum ramosum, C. scindens, C. hiranonsis, C. bifermentans, C.leptum and C. sardiniensis.

In one embodiment, a minimal microbial consortium consisting essentiallyof: Clostridum ramosum, C. scindens, C. hiranonsis, C. bifermentans, C.leptum and C. sardiniensis is used in the prevention and/or treatment ofexisting allergic reactions to food.

In one embodiment, a minimal microbial consortium consists essentiallyof: Bacteroides fragilis, B. thetaiotaomicron, B. ovatus,Parabacteroides goldsteinii, and Prevotella tannerae.

In one embodiment, a minimal microbial consortium consisting essentiallyof: Bacteroides fragilis, B. thetaiotaomicron, B. ovatus,Parabacteroides goldsteinii, and Prevotella tannerae is used to treatexisting allergic reactions to food.

By “consists essentially of” in this context is meant that if theaddition of another microbe does not improve the treatment or preventionof allergy as described and defined herein, that microbe is notessential to the protective or therapeutic effect.

Prebiotics

A prebiotic is a selectively fermented ingredient that allows specificchanges, both in the composition and/or activity in the gastrointestinalmicrobiota, that confers neutral or positive benefits upon hostwell-being and health. Prebiotics can include complex carbohydrates,amino acids, peptides, or other nutritional components useful for thesurvival, colonization and persistence of the bacterial composition.Prebiotics include, but are not limited to, amino acids, biotin,fructooligosaccharide, galactooligosaccharides, inulin, lactulose,mannan oligosaccharides, oligofructose-enriched inulin, oligofructose,oligodextrose, tagatose, trans-galactooligosaccharide, andxylooligosaccharides.

Suitable prebiotics are usually plant-derived complex carbohydrates,oligosaccharides or polysaccharides. Generally, prebiotics areindigestible or poorly digested by humans and serve as a food source forbacteria. Prebiotics, which can be used in the pharmaceutical dosageforms, and pharmaceutical compositions provided herein include, withoutlimitation, galactooligosaccharides (GOS),trans-galactooligosaccharides, fructooligosaccharides or oligofructose(FOS), inulin, oligofructose-enriched inulin, lactulose, arabinoxylan,xylooligosaccharides (XOS), mannooligosaccharides, gum guar, gum Arabic,tagatose, amylose, amylopectin, xylan, pectin, and the like andcombinations of thereof. Prebiotics can be found in certain foods, e.g.,chicory root, Jerusalem artichoke, Dandelion greens, garlic, leek,onion, asparagus, wheat bran, wheat flour, banana, milk, yogurt,sorghum, burdock, broccoli, Brussels sprouts, cabbage, cauliflower,collard greens, kale, radish and rutabaga, and miso. Alternatively,prebiotics can be purified or chemically or enzymatically synthesized.

In some embodiments, the composition comprises at least one prebiotic.In one embodiment, the prebiotic is a carbohydrate. In some embodiments,the composition comprises a prebiotic mixture, which comprises at leastone carbohydrate. A “carbohydrate” refers to a sugar or polymer ofsugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and“oligosaccharide” can be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula (CH₂O)n. A carbohydrate can be a monosaccharide, a disaccharide,trisaccharide, oligosaccharide, or polysaccharide. The most basiccarbohydrate is a monosaccharide, such as glucose, sucrose, galactose,mannose, ribose, arabinose, xylose, and fructose. Disaccharides are twojoined monosaccharides. Exemplary disaccharides include sucrose,maltose, cellobiose, and lactose. Typically, an oligosaccharide includesbetween three and six monosaccharide units (e.g., raffinose, stachyose),and polysaccharides include six or more monosaccharide units. Exemplarypolysaccharides include starch, glycogen, and cellulose. Carbohydratescan contain modified saccharide units, such as 2′-deoxyribose wherein ahydroxyl group is removed, 2′-fluororibose wherein a hydroxyl group isreplace with a fluorine, or N-acetylglucosamine, a nitrogen-containingform of glucose (e.g., 2′-fluororibose, deoxyribose, and hexose).Carbohydrates can exist in many different forms, for example,conformers, cyclic forms, acyclic forms, stereoisomers, tautomers,anomers, and isomers. Carbohydrates can be purified from natural (e.g.,plant or microbial) sources (i.e., they are enzymatically synthesized),or they can be chemically synthesized or modified.

Suitable prebiotic carbohydrates can include one or more of acarbohydrate, carbohydrate monomer, carbohydrate oligomer, orcarbohydrate polymer. In certain embodiments, the pharmaceuticalcomposition or dosage form comprises at least one type of microbe and atleast one type of non-digestible saccharide, which includesnon-digestible monosaccharides, non-digestible oligosaccharides, ornon-digestible polysaccharides. In one embodiment, the sugar units of anoligosaccharide or polysaccharide can be linked in a single straightchain or can be a chain with one or more side branches. The length ofthe oligosaccharide or polysaccharide can vary from source to source. Inone embodiment, small amounts of glucose can also be contained in thechain. In another embodiment, the prebiotic composition can be partiallyhydrolyzed or contain individual sugar moieties that are components ofthe primary oligosaccharide (see e.g., U.S. Pat. No. 8,486,668).

Prebiotic carbohydrates can include, but are not limited tomonosaccharides (e.g., trioses, tetroses, pentoses, aldopentoses,ketopentoses, hexoses, cyclic hemiacetals, ketohexoses, heptoses) andmultimers thereof, as well as epimers, cyclic isomers, stereoisomers,and anomers thereof. Non-limiting examples of monosaccharides include(in either the L- or D-conformation) glyceraldehyde, threose, ribose,altrose, glucose, mannose, talose, galactose, gulose, idose, lyxose,arabanose, xylose, allose, erythrose, erythrulose, tagalose, sorbose,ribulose, psicose, xylulose, fructose, dihydroxyacetone, and cyclic(alpha or beta) forms thereof. Multimers (disaccharides, trisaccharides,oligosaccharides, polysaccharides) thereof include, but are not limitedto, sucrose, lactose, maltose, lactulose, trehalose, cellobiose,kojibiose, nigerose, isomaltose, sophorose, laminaribiose, gentioboise,turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiulose,rutinose, rutinulose, xylobiose, primeverose, amylose, amylopectin,starch (including resistant starch), chitin, cellulose, agar, agarose,xylan, glycogen, bacterial polysaccharides such as capsularpolysaccharides, LPS, and peptidoglycan, and biofilm exopolysaccharide(e.g., alginate, EPS), N-linked glycans, and O-linked glycans. Prebioticsugars can be modified and carbohydrate derivatives include amino sugars(e.g., sialic acid, N-acetylglucosamine, galactosamine), deoxy sugars(e.g., rhamnose, fucose, deoxyribose), sugar phosphates, glycosylamines,sugar alcohols, and acidic sugars (e.g., glucuronic acid, ascorbicacid).

In one embodiment, the prebiotic carbohydrate component of thepharmaceutical composition consists essentially of one or morenon-digestible saccharides.

In one embodiment, the prebiotic carbohydrate component of thepharmaceutical composition allows the commensal colonic microbiota,comprising microorganisms associated with a healthy-state microbiome orpresenting a low risk of a patient developing an autoimmune orinflammatory condition, to be regularly maintained. In one embodiment,the prebiotic carbohydrate allows the co-administered or co-formulatedmicrobe or microbes to engraft, grow, and/or be regularly maintained ina mammalian subject. In some embodiments, the mammalian subject is ahuman subject, for example, a human subject having or suspected ofhaving a food allergy.

In some embodiments, the prebiotic favors the growth of an administeredmicrobe, wherein the growth of the administered microbe and/or thefermentation of the administered prebiotic by the administered microbeslows or reduces the growth of a pathogen or pathobiont. For example,FOS, neosugar, or inulin promotes the growth of acid-forming bacteria inthe colon such as bacteria belonging to the genus Lactobacillus orBifidobacterium and Lactobacillus acidophilus and Bifidobacteriumbifidus can play a role in reducing the number of pathogenic bacteria inthe colon (see e.g., U.S. Pat. No. 8,486,668). Other polymers, such asvarious galactans, lactulose, and carbohydrate based gums, such aspsyllium, guar, carrageen, gellan, and konjac, are also known to improvegastrointestinal (GI) health.

In some embodiments, the prebiotic comprises one or more of GOS,lactulose, raffinose, stachyose, lactosucrose, FOS (i.e., oligofructoseor oligofructan), inulin, isomalto-oligosaccharide,xylo-oligosaccharide, paratinose oligosaccharide, transgalactosylatedoligosaccharides (i.e., transgalacto-oligosaccharides),transgalactosylate disaccharides, soybean oligosaccharides (i.e.,soyoligosaccharides), gentiooligosaccharides, glucooligosaccharides,pecticoligosaccharides, palatinose polycondensates, difructose anhydrideIII, sorbitol, maltitol, lactitol, polyols, polydextrose, reducedparatinose, cellulose, β-glucose, β-galactose, β-fructose, verbascose,galactinol, and β-glucan, guar gum, pectin, high, sodium alginate, andlambda carrageenan, or mixtures thereof. The GOS may be a short-chainGOS, a long-chain GOS, or any combination thereof. The FOS can be ashort-chain FOS, a long-chain FOS, or any combination thereof.

In some embodiments, the prebiotic composition comprises twocarbohydrate species (non-limiting examples being a GOS and FOS) in amixture of at least 1:1, at least 2:1, at least 5:1, at least 9:1, atleast 10:1, about 20:1, or at least 20:1.

In some embodiments, the prebiotic comprises a mixture of one or morenon-digestible oligosaccharides, non-digestible polysaccharides, freemonosaccharides, non-digestible saccharides, starch, or non-starchpolysaccharides.

Oligosaccharides are generally considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. Most oligosaccharides described herein aredescribed with the name or abbreviation for the non-reducing saccharide(e.g., Gal or D-Gal), preceded or followed by the configuration of theglycosidic bond (a or (3), the ring bond, the ring position of thereducing saccharide involved in the bond, and then the name orabbreviation of the reducing saccharide (e.g., Glc or D-Glc). Thelinkage (e.g., glycosidic linkage, galactosidic linkage, glucosidiclinkage) between two sugar units can be expressed, for example, as 1,4,1->4, or (1-4).

Both FOS and GOS are non-digestible saccharides. β glycosidic linkagesof saccharides, such as those found in, but not limited to, FOS and GOS,make these prebiotics mainly non-digestible and unabsorbable in thestomach and small intestine α-linked GOS (α-GOS) is also not hydrolyzedby human salivary amylase, but can be used by Bifidobacterium bifidumand Clostridium butyricum (Yamashita A. et al., 2004. J. Appl. Glycosci.51:115-122). FOS and GOS can pass through the small intestine and intothe large intestine (colon) mostly intact, except where commensalmicrobes and microbes administered as part of a pharmaceuticalcomposition are able to metabolize the oligosaccharides.

GOS (also known as galacto-oligosaccharides, galactooligosaccharides,trans-oligosaccharide (TOS), trans-galacto-oligosaccharide (TGOS), andtrans-galactooligosaccharide) are oligomers or polymers of galactosemolecules ending mainly with a glucose or sometimes ending with agalactose molecule and have varying degree of polymerization (generallythe DP is between 2-20) and type of linkages. In one embodiment, GOScomprises galactose and glucose molecules. In another embodiment, GOScomprises only galactose molecules. In a further embodiment, GOS aregalactose-containing oligosaccharides of the form of[β-D-Gal-(1-6)]_(n)-β-D-Gal-(1-4)-D-Glc wherein n is 2-20. In anotherembodiment, GOS are galactose-containing oligosaccharides of the formGlc α1-4-[β Gal 1-6)]_(n) where n=2-20. In another embodiment, GOS arein the form of α-D-Glc (1-4)-[β-D-Gal-(1-6)]_(n) where n=2-20. Gal is agalactopyranose unit and Glc (or Glu) is a glucopyranose unit.

In one embodiment, a prebiotic composition comprises a GOS-relatedcompound. A GOS-related compound can have the following properties: a) a“lactose” moiety; e.g., GOS with a gal-glu moiety and any polymerizationvalue or type of linkage; or b) be stimulatory to “lactose fermenting”microbes in the human GI tract; for example, raffinose (gal-fru-glu) isa “related” GOS compound that is stimulatory to both lactobacilli andbifidobacteria.

Linkages between the individual sugar units found in GOS and otheroligosaccharides include β-(1-6), β-(1-4), β-(1-3) and β-(1-2) linkages.In one embodiment, the administered oligosaccharides (e.g., GOS) arebranched saccharides. In another embodiment, the administeredoligosaccharides (e.g., GOS) are linear saccharides.

Alpha-GOS (also called alpha-bond GOS or alpha-linked GOS) areoligosaccharides having an alpha-galactopyranosyl group. Alpha-GOScomprises at least one alpha glycosidic linkage between the saccharideunits. Alpha-GOS are generally represented by α-(Gal)_(n) (n usuallyrepresents an integer of 2 to 10) or α-(Gal)_(n) Glc (n usuallyrepresents an integer of 1 to 9). Examples include a mixture ofα-galactosylglucose, α-galactobiose, α-galactotriose, α-galactotetraose,and higher oligosaccharides. Additional non-limiting examples includemelibiose, manninootriose, raffinose, stachyose, and the like, which canbe produced from beat, soybean oligosaccharide, and the like.

Commercially available and enzyme synthesized alpha-GOS products arealso useful for the compositions described herein. Synthesis ofalpha-GOS with an enzyme is conducted utilizing the dehydrationcondensation reaction of α-galactosidase with the use of galactose,galactose-containing substance, or glucose as a substrate. Thegalactose-containing substance includes hydrolysates ofgalactose-containing substances, for example, a mixture of galactose andglucose obtained by allowing beta-galactosidase to act on lactose, andthe like. Glucose can be mixed separately with galactose and be used asa substrate with α-galactosidase (see e.g., WO 02/18614). Methods ofpreparing alpha-GOS have been described (see e.g., EP 514551 andEP2027863).

In one embodiment, a GOS composition comprises a mixture of saccharidesthat are alpha-GOS and saccharides that are produced bytransgalactosylation using β-galactosidase. In another embodiment, GOScomprises alpha-GOS. In another embodiment, alpha-GOS comprises α-(Gal)₂from 10% to 100% by weight. In one embodiment, GOS comprises onlysaccharides that are produced by transgalactosylation usingβ-galactosidase.

In one embodiment, the pharmaceutical composition comprises, in additionto one or more microbes, an oligosaccharide composition that is amixture of oligosaccharides comprising 1-20% by weight ofdi-saccharides, 1-20% by weight tri-saccharides, 1-20% by weighttetra-saccharides, and 1-20% by weight penta-saccharides. In anotherembodiment, an oligosaccharide composition is a mixture ofoligosaccharides consisting essentially of 1-20% by weight ofdi-saccharides, 1-20% by weight tri-saccharides, 1-20% by weighttetra-saccharides, and 1-20% by weight penta-saccharides.

In one embodiment, a prebiotic composition is a mixture ofoligosaccharides comprising 1-20% by weight of saccharides with a degreeof polymerization (DP) of 1-3, 1-20% by weight of saccharides with DP of4-6, 1-20% by weight of saccharides with DP of 7-9, and 1-20% by weightof saccharides with DP of 10-12, 1-20% by weight of saccharides with DPof 13-15.

In another embodiment, a prebiotic composition comprises a mixture ofoligosaccharides comprising 50-55% by weight of di-saccharides, 20-30%by weight tri-saccharides, 10-20% by weight tetra-saccharide, and 1-10%by weight penta-saccharides. In one embodiment, a GOS composition is amixture of oligosaccharides comprising 52% by weight of di-saccharides,26% by weight tri-saccharides, 14% by weight tetra-saccharide, and 5% byweight penta-saccharides. In another embodiment, a prebiotic compositioncomprises a mixture of oligosaccharides comprising 45-55% by weighttri-saccharides, 15-25% by weight tetra-saccharides, 1-10% by weightpenta-saccharides.

In certain embodiments, the composition comprises a mixture of neutraland acid oligosaccharides as disclosed in e.g., WO 2005/039597 (N.V.Nutricia) and US Patent Application 20150004130. In one embodiment, theacid oligosaccharide has a degree of polymerization (DP) between 1 and5000. In another embodiment, the DP is between 1 and 1000. In anotherembodiment, the DP is between 2 and 250. If a mixture of acidoligosaccharides with different degrees of polymerization is used, theaverage DP of the acid oligosaccharide mixture is preferably between 2and 1000. The acid oligosaccharide can be a homogeneous or heterogeneouscarbohydrate. The acid oligosaccharides can be prepared from pectin,pectate, alginate, chondroitine, hyaluronic acids, heparin, heparane,bacterial carbohydrates, sialoglycans, fucoidan, fucooligosaccharides orcarrageenan, and are preferably prepared from pectin or alginate. Theacid oligosaccharides can be prepared by the methods described in e.g.,WO 01/60378, which is hereby incorporated by reference. The acidoligosaccharide is preferably prepared from high methoxylated pectin,which is characterized by a degree of methoxylation above 50%. As usedherein, “degree of methoxylation” (also referred to as DE or “degree ofesterification”) is intended to mean the extent to which free carboxylicacid groups contained in the polygalacturonic acid chain have beenesterified (e.g. by methylation). In some embodiments, the acidoligosaccharides have a degree of methoxylation above about 10%, aboveabout 20%, above about 50%, above about 70%. In some embodiments, theacid oligosaccharides have a degree of methylation above about 10%,above about 20%, above about 50%, above about 70%.

The term neutral oligosaccharides as used in the present inventionrefers to saccharides which have a degree of polymerization of monoseunits exceeding 2, exceeding 3, exceeding 4, or exceeding 10, which arenot or only partially digested in the intestine by the action of acidsor digestive enzymes present in the human upper digestive tract (smallintestine and stomach) but which are fermented by the human intestinalflora and preferably lack acidic groups. The neutral oligosaccharide isstructurally (chemically) different from the acid oligosaccharide. Theterm “neutral oligosaccharides”, as used herein, refers to saccharideswhich have a degree of polymerization of the oligosaccharide below 60monose units. The term “monose units” refers to units having a closedring structure e.g., the pyranose or furanose forms. In someembodiments, the neutral oligosaccharide comprises at least 90% or atleast 95% monose units selected from the group consisting of mannose,arabinose, fructose, fucose, rhamnose, galactose, -D-galactopyranose,ribose, glucose, xylose and derivatives thereof, calculated on the totalnumber of monose units contained therein. Suitable neutraloligosaccharides are preferably fermented by the gut flora. Non-limitingexamples of suitable neutral oligosaccharides are cellobiose(4-O-β-D-glucopyranosyl-D-glucose), cellodextrins((4-O-β-D-glucopyranosyl)n-D-glucose), β-cyclo-dextrins (Cyclicmolecules of α-1-4-linked D-glucose; α-cyclodextrin-hexamer,β-cyclodextrin-heptamer and γ-cyclodextrin-octamer), indigestibledextrin, gentiooligosaccharides (mixture of β-1-6 linked glucoseresidues, some 1-4 linkages), glucooligosaccharides (mixture ofα-D-glucose), isomaltooligosaccharides (linear α-1-6 linked glucoseresidues with some 1-4 linkages), isomaltose(6-O-α-D-glucopyranosyl-D-glucose); isomaltriose(6-O-α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyl-D-glucose), panose(6-O-α-D-glucopyranosyl-(1-6)-α-D-glucopyranosyl-(1-4)-D-glucose),leucrose (5-O-α-D-glucopyranosyl-D-fructopyranoside), palatinose orisomaltulose (6-O-α-D-glucopyranosyl-D-fructose), theanderose(O-α-D-glucopyranosyl-(1-6)-O-α-D-glucopyranosyl-(1-2)-β-D-fructofuranoside),D-agatose, D-lyxo-hexylose, lactosucrose(O-β-D-galactopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-2)-β-D-fructofuranoside),α-galactooligosaccharides including raffinose, stachyose and other soyoligosaccharides(O-α-D-galactopyranosyl-(1-6)-α-D-glucopyranosyl-β-D-fructofuranoside),β-galactooligosaccharides or transgalacto-oligosaccharides(β-D-galactopyranosyl-(1-6)-[β-D-glucopyranosyl]n-(1-4) α-D glucose),lactulose (4-O-β-D-galactopyranosyl-D-fructose), 4′-galatosyllactose(β-D-galactopyranosyl-(1-4)-O-β-D-glucopyranosyl-(1-4)-D-glucopyranose),synthetic galactooligosaccharide (neogalactobiose, isogalactobiose,galsucrose, isolactose I, II and III), fructans-Levan-type(β-D-(2→6)-fructofuranosyl)n α-D-glucopyranoside), fructans-Inulin-type(β-D-((2→1)-fructofuranosyl)n α-D-glucopyranoside), 1f-β-fructofuranosylnystose (β-D-((2→1)-fructofuranosyl)nB-D-fructofuranoside), xylooligo-saccharides (B-D-((1→4)-xylose)n,lafinose, lactosucrose and arabinooligosaccharides.

In some embodiments, the neutral oligosaccharide is selected from thegroup consisting of fructans, fructooligosaccharides, indigestibledextrins galactooligo-saccharides (includingtransgalactooligosaccharides), xylooligosaccharides,arabinooligo-saccharides, glucooligosaccharides, mannooligosaccharides,fucooligosaccharides and mixtures thereof.

Suitable oligosaccharides and their production methods are furtherdescribed in Laere K. J. M. (Laere, K. J. M., Degradation ofstructurally different non-digestible oligosaccharides by intestinalbacteria: glycosylhydrolases of Bi, adolescentis. PhD-thesis (2000),Wageningen Agricultural University, Wageningen, The Netherlands), theentire content of which is hereby incorporated by reference.Transgalactooligosaccharides (TOS) are for example sold under thetrademark Vivinal™ (Borculo Domo Ingredients, Netherlands). Indigestibledextrin, which can be produced by pyrolysis of corn starch, comprisesα(1→4) and α(1→6) glucosidic bonds, as are present in the native starch,and contains 1→2 and 1→3 linkages and levoglucosan. Due to thesestructural characteristics, indigestible dextrin containswell-developed, branched particles that are partially hydrolyzed byhuman digestive enzymes. Numerous other commercial sources ofindigestible oligosaccharides are readily available and known to skilledpersons in the art. For example, transgalactooligosaccharide isavailable from Yakult Honsha Co., Tokyo, Japan. Soybean oligosaccharideis available from Calpis Corporation distributed by Ajinomoto U.S.A.Inc., Teaneck, N.J.

In a further embodiment, the prebiotic mixture of the pharmaceuticalcomposition described herein comprises an acid oligosaccharide with a DPbetween 1 and 5000, prepared from pectin, alginate, and mixturesthereof; and a neutral oligosaccharide, selected from the group offructans, fructooligosaccharides, indigestible dextrins,galactooligosaccharides including transgalacto-oligosaccharides,xylooligosaccharides, arabinooligosaccharides, glucooligosaccharides,mannooligosaccharides, fucooligosaccharides, and mixtures thereof.

In certain embodiments, the prebiotic mixture comprises xylose. In otherembodiments, the prebiotic mixture comprises a xylose polymer (i.e.xylan). In some embodiments, the prebiotic comprises xylose derivatives,such as xylitol, a sugar alcohol generated by reduction of xylose bycatalytic hydrogenation of xylose, and also xylose oligomers (e.g.,xylooligosaccharide). While xylose can be digested by humans, viaxylosyltransferase activity, most xylose ingested by humans is excretedin urine. In contrast, some microorganisms are efficient at xylosemetabolism or can be selected for enhanced xylose metabolism. Microbialxylose metabolism can occur by at least four pathways, including theisomerase pathway, the Weimburg pathway, the Dahms pathway, and, foreukaryotic microorganisms, the oxido-reductase pathway.

The xylose isomerase pathway involves the direct conversion of D-xyloseinto D-xylulose by xylose isomerase, after which D-xylulose isphosphorylated by xylulose kinase to yield D-xylolose-5-phosphate, anintermediate of the pentose phosphate pathway.

In the Weimberg pathway, D-xylose is oxidized to D-xylono-lactone by aD-xylose dehydrogenase. Then D-xylose dehydrogenase is hydrolyzed by alactonase to yield D-xylonic acid, and xylonate dehydratase activitythen yields 2-keto-3-deoxy-xylonate. The final steps of the Weimbergpathway are a dehydratase reaction to form 2-keto glutarate semialdehydeand an oxidizing reaction to form 2-ketoglutarate, an intermediate ofthe Krebs cycle.

The Dahms pathway follows the same mechanism as the Weimberg pathway butdiverges once it has yielded 2-keto-3-deoxy-xylonate. In the Dahmspathway, an aldolase splits 2-keto-3-deoxy-xylonate into pyruvate andglycolaldehyde.

The xylose oxido-reductase pathway, also known as the xylosereductase-xylitol dehydrogenase pathway, begins by the reduction ofD-xylose to xylitol by xylose reductase followed by the oxidation ofxylitol to D-xylulose by xylitol dehydrogenase. As in the isomerasepathway, the next step in the oxido-reductase pathway is thephosphorylation of D-xylulose by xylulose kinase to yieldD-xylolose-5-phosphate.

Xylose is present in foods like fruits and vegetables and other plantssuch as trees for wood and pulp production. Thus, xylose can be obtainedin the extracts of such plants. Xylose can be obtained from variousplant sources using known processes including acid hydrolysis followedby various types of chromatography. Examples of such methods to producexylose include those described in Maurelli, L. et al. (2013), Appl.Biochem. Biotechnol. 170:1104-1118; Hooi H. T et al. (2013), Appl.Biochem. Biotechnol. 170:1602-1613; Zhang H-J. et al. (2014), BioprocessBiosyst. Eng. 37:2425-2436.

Culture and Storage of Consortium Constituents

For banking, the strains included in the bacterial composition can be(1) isolated directly from a specimen or taken from a banked stock, (2)optionally cultured on a nutrient agar or broth that supports growth togenerate viable biomass, and (3) the biomass optionally preserved inmultiple aliquots in long-term storage.

In embodiments using a culturing step, the agar or broth containsnutrients that provide essential elements and specific factors thatenable growth. An example would be a medium composed of 20 g/L glucose,10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/Lsodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/Lmagnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, and1 mg/L menadione. A variety of microbiological media and variations arewell known in the art (e.g. R. M. Atlas, Handbook of MicrobiologicalMedia (2010) CRC Press). Medium can be added to the culture at thestart, can be added during the culture, or can beintermittently/continuously flowed through the culture. The strains inthe bacterial composition can be cultivated alone, as a subset of thebacterial composition, or as an entire collection comprising thebacterial composition. As an example, a first strain can be cultivatedtogether with a second strain in a mixed continuous culture, at adilution rate lower than the maximum growth rate of either cell toprevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for atime sufficient to build biomass. For bacterial compositions for humanuse this is often at normal body temperature (37° C.), pH, and otherparameter with values similar to the normal human niche. The environmentcan be actively controlled, passively controlled (e.g., via buffers), orallowed to drift. For example, for anaerobic bacterial compositions(e.g., gut microbiota), an anoxic/reducing environment can be employed.This can be accomplished by addition of reducing agents/factors such ascysteine to the broth, and/or stripping it of oxygen. As an example, aculture of a bacterial composition can be grown at 37° C., pH 7, in themedium above, pre-reduced with 1 g/L cysteine.HCl.

When the culture has generated sufficient biomass, it can be preservedfor banking or storage. The organisms can be placed into a chemicalmilieu that protects from freezing (adding ‘cryoprotectants’), drying(‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensinginto multiple (optionally identical) containers to create a uniformbank, and then treating the culture for preservation. Containers aregenerally impermeable and have closures that assure isolation from theenvironment. Cryopreservation treatment is accomplished by freezing aliquid at ultra-low temperatures (e.g., at or below −80° C.). Driedpreservation removes water from the culture by evaporation (in the caseof spray drying or ‘cool drying’) or by sublimation (e.g., for freezedrying, spray freeze drying). Removal of water improves long-termbacterial composition storage stability at temperatures elevated abovecryogenic. If the bacterial composition comprises spore forming speciesand results in the production of spores, the final composition can bepurified by additional means such as density gradient centrifugationpreserved using the techniques described above. Bacterial compositionbanking can be done by culturing and preserving the strainsindividually, or by mixing the strains together to create a combinedbank. As an example of cryopreservation, a bacterial composition culturecan be harvested by centrifugation to pellet the cells from the culturemedium, the supernate decanted and replaced with fresh culture brothcontaining 15% glycerol. The culture can then be aliquoted into 1 mLcryotubes, sealed, and placed at −80° C. for long-term viabilityretention. This procedure achieves acceptable viability upon recoveryfrom frozen storage.

Organism production can be conducted using similar culture steps tobanking, including medium composition and culture conditions. It can beconducted at larger scales of operation, especially for clinicaldevelopment or commercial production. At larger scales, there can beseveral subcultivations of the bacterial composition prior to the finalcultivation. At the end of cultivation, the culture is harvested toenable further formulation into a dosage form for administration. Thiscan involve concentration, removal of undesirable medium components,and/or introduction into a chemical milieu that preserves the bacterialcomposition and renders it acceptable for administration via the chosenroute. For example, a bacterial composition can be cultivated to aconcentration of 10¹⁰ CFU/mL, then concentrated 20-fold by tangentialflow microfiltration; the spent medium may be exchanged by diafilteringwith a preservative medium consisting of 2% gelatin, 100 mM trehalose,and 10 mM sodium phosphate buffer. The suspension can then befreeze-dried to a powder and titrated.

After drying, the powder can be blended to an appropriate potency, andmixed with other cultures and/or a filler such as microcrystallinecellulose for consistency and ease of handling, and the bacterialcomposition formulated as provided herein.

In one embodiment, a composition comprising a microbial consortium asdescribed herein, is not a fecal transplant. In some embodiments all oressentially all of the bacterial entities present in a purifiedpopulation are originally obtained from a fecal material andsubsequently, e.g., for production of pharmaceutical compositions, aregrown in culture as described herein or otherwise known in the art. Inone embodiment, the bacterial cells are cultured from a bacterial stockand purified as described herein. In one embodiment, each of thepopulations of bacterial cells are independently cultured and purified,e.g., each population is cultured separately and subsequently mixedtogether. In one embodiment, one or more of the populations of bacterialcells in the composition are co-cultured.

Dosage, Administration and Formulations

In some embodiments, cells over a range of, for example, 2-5×10⁵, ormore, e.g., 1×10⁶, 1×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰ ormore can be administered in a composition comprising a microbialconsortium. The dosage range for the bacteria depends upon the potency,and include amounts large enough to produce the desired effect, e.g.,reduction in at least one symptom of a food allergy in a treatedsubject. The dosage should not be so large as to cause unacceptableadverse side effects. Generally, the dosage will vary with the type ofillness, and with the age, condition, and sex of the patient. The dosagecan be determined by one of skill in the art and can also be adjusted bythe individual physician in the event of any complication.

For use in the various aspects described herein, an effective amount ofcells in a composition as described herein comprises at least 10²bacterial cells, at least 1×10³ bacterial cells, at least 1×10⁴bacterial cells, at least 1×10⁵ bacterial cells, at least 1×10⁶bacterial cells, at least 1×10⁷ bacterial cells, at least 1×10⁸bacterial cells, at least 1×10⁹ bacterial cells, at least 1×10¹⁰bacterial cells, at least 1×10¹¹ bacterial cells, at least 1×10¹²bacterial cells or more. Where a microbial consortium is isolated and/orpurified from a subject that is tolerant to a selected food allergen,the bacterial cells can be derived from one or more donors, or can beobtained from an autologous source. In some embodiments of the aspectsdescribed herein, the cells of the microbial consortium are expanded ormaintained in culture prior to administration to a subject in needthereof. In one embodiment, the microbial consortium is obtained from amicrobe bank. Members of a therapeutic or preventive/prophylacticconsortium are generally administered together, e.g., in a singleadmixture. However, it is specifically contemplated herein that membersof a given consortium can be administered as separate dosage forms orsub-mixtures or sub-combinations of the consortium members. Thus, for aconsortium of e.g., six members, the consortium can be administered, forexample, as a single preparation including all six members (in one ormore dosage units, e.g., one or more capsules) or as two or moreseparate preparations that, in sum, include all members of the givenconsortium. While administration as a single admixture is preferred, apotential advantage of the use of e.g., individual units for each memberof a consortium, is that the actual strains administered to any givensubject can be tailored, if necessary, by selecting the appropriatecombination of, for example, single species dosage units that togethercomprise the desired consortium.

Biomass of Administered Species, Per Dose, Vs. Known In Vivo Biomass:

It is contemplated herein that the consortium composition is formulatedto deliver a larger biomass than the normal biomass of the commensalorganisms in a “healthy” individual. For example, the range of biomassescontemplated for delivery and colonization can be found in Table 1,column 2, as compared to the normal biomass in a healthy individual asshown in Table 1, columns 3 & 4. The table below shows the range ofadministered biomasses of organisms relative to published data atspecific locations. Note, in many cases the bacterial quantitation inGustafsson, 1982 was to general categories of organisms, such asClostridia, and incorporated multiple species under those headers.Individual species in the consortia would thus likely be less than theactual highest reported biomass at the specific locations; the small andlarge intestinal biomass data should thus be considered an upper-boundfor what might occur in vivo in normal individuals.

Small Large Consortia Intestinal Intestinal Species Biomass BiomassBiomass Reference Bacteroides 1 × 10⁷- <10³ CFU/g in 10⁸-10¹¹Gustafsson, fragilis 5 × 10⁸ duodenum- CFU/g 1982. [1] CFU/mL jejunum10³-10⁸ CFU/g in ileum B. 1 × 10⁷- <10³ CFU/g in 10⁸-10¹¹ Gustafsson,thetaiotaomicron 5 × 10⁸ duodenum- CFU/g 1982. CFU/mL jejunum Bry, 1996.[2] 10³-10⁸ CFU/g in ileum B. ovatus 1 × 10⁷- <10³ CFU/g in 10⁸-10¹¹Gustafsson, 5 × 10⁸ duodenum- CFU/g 1982. CFU/mL jejunum 10³-10⁸ CFU/gin ileum C. bifermentans 1 × 10⁷- <10³ CFU/g in  0-10⁶ Gustafsson, 5 ×10⁸ duodenum- CFU/g 1982. CFU/mL jejunum 10²-10⁴ CFU/g in ileum C.hiranonsis 1 × 10⁷- <10³ CFU/g in  0-10⁶ Gustafsson, 5 × 10⁸ duodenum-CFU/g 1982. CFU/mL jejunum 10²-10⁴ CFU/g in ileum C. leptum 1 × 10⁷-<10³ CFU/g in  0-10⁶ Gustafsson, 5 × 10⁸ duodenum- CFU/g 1982. CFU/mLjejunum 10²-10⁴ CFU/g in ileum Clostridium 1 × 10⁷- <10³ CFU/g in  0-10⁶Gustafsson, ramosum 5 × 10⁸ duodenum- CFU/g 1982. CFU/mL jejunum 10²-10⁴CFU/g in ileum C. sardiniensis 1 × 10⁷- <10³ CFU/g in  0-10⁶ Gustafsson,5 × 10⁸ duodenum- CFU/g 1982. CFU/mL jejunum 10²-10⁴ CFU/g in ileum C.scindens 1 × 10⁷- <10³ CFU/g in  0-10⁶ Gustafsson, 5 × 10⁸ duodenum-CFU/g 1982. CFU/mL jejunum 10²-10⁴ CFU/g in ileum Parabacteroides 1 ×10⁷- <10³ CFU/g in  0-10⁶ Gustafsson, goldsteinii 5 × 10⁸ duodenum-CFU/g 1982. CFU/mL jejunum 10³-10⁸ CFU/g in ileum Prevotella 1 × 10⁷-<10³ CFU/g in  0-10⁶ Gustafsson, tannerae 5 × 10⁸ duodenum- CFU/g 1982.CFU/mL jejunum <10⁴ CFU/g in ileum References: 1. Gustafsson, B E. Thephysiological importance of the colonic microflora. Scand J.Gastroenterol Suppl. 1982, 77: 117-31. 2. Bry L, et al. A model ofhost-microbial interactions in an open mammalian ecosystem. Science.1996, 273(5280): 1380-3.

A pharmaceutical composition comprising a microbial consortium can beadministered by any method suitable for depositing in thegastrointestinal tract, preferably the colon, of a subject (e.g., human,mammal, animal, etc.). Examples of routes of administration includerectal administration by colonoscopy, suppository, enema, upperendoscopy, or upper push enteroscopy. Additionally, intubation throughthe nose or the mouth by nasogastric tube, nasoenteric tube, or nasaljejunal tube can be utilized. Oral administration by a solid such as apill, tablet, a suspension, a gel, a geltab, a semisolid, a tablet, asachet, a lozenge or a capsule or microcapsule, or as an enteralformulation, or re-formulated for final delivery as a liquid, asuspension, a gel, a geltab, a semisolid, a tablet, a sachet, a lozengeor a capsule, or as an enteral formulation can be utilized as well. Alsocontemplated herein are food items that are inoculated with a microbialconsortium as described herein. Compositions can also be treated oruntreated fecal flora, entire (or substantially entire) microbiota, orpartially, substantially or completely isolated or purified fecal flora,and can be lyophilized, freeze-dried or frozen, or processed into apowder.

In some embodiments, the compositions described herein can beadministered in a form containing one or more pharmaceuticallyacceptable carriers. Suitable carriers are well known in the art andvary with the desired form and mode of administration of thecomposition. For example, pharmaceutically acceptable carriers caninclude diluents or excipients such as fillers, binders, wetting agents,disintegrators, surface-active agents, glidants, lubricants, and thelike. Typically, the carrier may be a solid (including powder), liquid,or combinations thereof. Each carrier is preferably “acceptable” in thesense of being compatible with the other ingredients in the compositionand not injurious to the subject. The carrier may be biologicallyacceptable and inert (e.g., it permits the composition to maintainviability of the biological material until delivered to the appropriatesite).

Oral compositions can include an inert diluent or an edible carrier. Forthe purpose of oral therapeutic administration, the active compound canbe incorporated with excipients and used in the form of tablets,lozenges, pastilles, troches, or capsules, e.g., gelatin capsules. Oralcompositions can also be prepared by combining a composition of thepresent disclosure with a food. In one embodiment a food used foradministration is chilled, for instance, iced flavored water. In certainembodiments, the food item is not a potentially allergenic food item(e.g., not soy, wheat, peanut, tree nuts, dairy, eggs, shellfish orfish). Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, orange flavoring, or other suitableflavorings. These are for purposes of example only and are not intendedto be limiting.

The compositions comprising a microbial consortium can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery. The compositions can be prepared with carriers thatwill protect the consortium against rapid elimination from the body,such as a controlled release formulation, including implants.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Such formulations can be preparedusing standard techniques. The materials can also be obtainedcommercially from, for instance, Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art.

In some embodiments, a composition can be encapsulated (e.g.,enteric-coated formulations). For instance, when the composition is tobe administered orally, the dosage form is formulated so the compositionis not exposed to conditions prevalent in the gastrointestinal tractbefore the small intestine, e.g., high acidity and digestive enzymespresent in the stomach. The encapsulation of compositions fortherapeutic use is routine in the art. Encapsulation can includehard-shelled capsules, which can be used for dry, powdered ingredientssoft-shelled capsules. Capsules can be made from aqueous solutions ofgelling agents such as animal protein (e.g., gelatin), plantpolysaccharides or derivatives like carrageenans and modified forms ofstarch and cellulose. Other ingredients can be added to a gelling agentsolution such as plasticizers (e.g., glycerin and or sorbitol), coloringagents, preservatives, disintegrants, lubricants and surface treatment.

In one embodiment, a microbial consortium as described herein isformulated with an enteric coating. An enteric coating can control thelocation of where a microbial consortium is released in the digestivesystem. Thus, an enteric coating can be used such that a microbialconsortium-containing composition does not dissolve and release themicrobes in the stomach, which can be a toxic environment for manymicrobes, but rather travels to the small intestine, where it dissolvesand releases the microbes in an environment where they can survive. Anenteric coating can be stable at low pH (such as in the stomach) and candissolve at higher pH (for example, in the small intestine). Materialthat can be used in enteric coatings includes, for example, alginicacid, cellulose acetate phthalate, plastics, waxes, shellac, and fattyacids (e.g., stearic acid, palmitic acid). Enteric coatings aredescribed, for example, in U.S. Pat. Nos. 5,225,202, 5,733,575,6,139,875, 6,420,473, 6,455,052, and 6,569,457, all of which are hereinincorporated by reference in their entirety. The enteric coating can bean aqueous enteric coating. Examples of polymers that can be used inenteric coatings include, for example, shellac (trade name EmCoat 120 N,Marcoat 125); cellulose acetate phthalate (trade names AQUACOAT™,AQUACOAT ECD™, SEPIFILM™, KLUCEL™, and METOLOSE™); polyvinylacetatephthalate (trade name SURETERIC™); and methacrylic acid (trade nameEUDRAGIT™).

In one embodiment, an enteric coated probiotic composition comprisingmembers of a microbial consortium as described herein is administered toa subject. In another embodiment, an enteric coated probiotic andprebiotic composition is administered to a subject.

Formulations suitable for rectal administration include gels, creams,lotions, aqueous or oily suspensions, dispersible powders or granules,emulsions, dissolvable solid materials, douches, and the like. Theformulations are preferably provided as unit-dose suppositoriescomprising the active ingredient in one or more solid carriers formingthe suppository base, for example, cocoa butter. Suitable carriers forsuch formulations include petroleum jelly, lanolin, polyethyleneglycols,alcohols, and combinations thereof. Alternatively, colonic washes withthe rapid recolonization deployment agent of the present disclosure canbe formulated for colonic or rectal administration.

Formulations suitable for oral administration may be provided asdiscrete units, such as tablets, capsules, cachets, syrups, elixirs,prepared food items, microemulsions, solutions, suspensions, lozenges,or gel-coated ampules, each containing a predetermined amount of theactive compound; as powders or granules; as solutions or suspensions inaqueous or non-aqueous liquids; or as oil-in-water or water-in-oilemulsions.

In some embodiments, the microbial consortium can be formulated in afood item. Some non-limiting examples of food items to be used with themethods and compositions described herein include: popsicles, cheeses,creams, chocolates, milk, meat, drinks, yogurt, pickled vegetables,kefir, miso, sauerkraut, etc. In other embodiments, the food items canbe juices, refreshing beverages, tea beverages, drink preparations,jelly beverages, and functional beverages; alcoholic beverages such asbeers; carbohydrate-containing foods such as rice food products,noodles, breads, and pastas; paste products such as fish, hams,sausages, paste products of seafood; retort pouch products such ascurries, food dressed with a thick starchy sauce, and Chinese soups;soups; dairy products such as milk, dairy beverages, ice creams,cheeses, and yogurts; fermented products such as fermented soybeanpastes, fermented beverages, and pickles; bean products; variousconfectionery products including biscuits, cookies, and the like,candies, chewing gums, gummies, cold desserts including jellies, creamcaramels, and frozen desserts; instant foods such as instant soups andinstant soy-bean soups; and the like. It is preferred that foodpreparations not require cooking after admixture with the microbialconsortium to avoid killing the microbes.

Formulations of a microbial consortium can be prepared by any suitablemethod, typically by uniformly and intimately admixing the consortiumwith liquids or finely divided solid carriers or both, in the requiredproportions and then, if necessary, shaping the resulting in mixtureinto the desired shape. In addition, the microbial consortium can betreated to prolong shelf-life, preferably the shelf-life of thepre-determined gut flora will be extended via freeze drying.

In some embodiments, the microbial consortium as described herein iscombined with one or more additional probiotic organisms prior totreatment of a subject. As used herein, the term “probiotic” refers tomicroorganisms that form at least a part of the transient or endogenousflora or microbial consortium and thereby exhibit a beneficialprophylactic and/or therapeutic effect on the host organism. Probioticsare generally known to be clinically safe (i.e., non-pathogenic) bythose individuals skilled in the art. Typical lactic acid-producingbacteria useful as a probiotic of this invention are efficient lacticacid producers which include non-pathogenic members of the Bacillusgenus which produce bacteriocins or other compounds which inhibit thegrowth of pathogenic organisms.

Exemplary lactic acid-producing, non-pathogenic Bacillus speciesinclude, but are not limited to: Bacillus coagulans; Bacillus coagulansHammer; and Bacillus brevis subspecies coagulans.

Exemplary lactic acid-producing Lactobacillus species include, but arenot limited to: Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus DDS-1, Lactobacillus GG, Lactobacillus rhamnosus,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus gasserii,Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus,bulgaricus, Lactobacillus salivarius and Lactobacillus sporogenes (alsodesignated as Bacillus coagulans). Exemplary lactic acid-producingSporolactobacillus species include all Sporolactobacillus species, forexample, Sporolactobacillus P44.

Exemplary lactic acid-producing Bifidiobacterium species include, butare not limited to: Bifidiobacterium adolescentis, Bifidiobacteriumanimalis, Bifidiobacterium bifidum, Bifidiobacterium bifidus,Bifidiobacterium breve, Bifidiobacterium infantis, Bifidiobacteriuminfantus, Bifidiobacterium longum, and any genetic variants thereof.

Examples of suitable non-lactic acid-producing Bacillus include, but arenot limited to: Bacillus subtilis, Bacillus uniflagellatus, Bacilluslateropsorus, Bacillus laterosporus BOD, Bacillus megaterium, Bacilluspolymyxa, Bacillus licheniformis, Bacillus pumilus, and Bacillussterothermophilus. Other strains that could be employed due to probioticactivity include members of the Streptococcus (Enterococcus) genus. Forexample, Enterococcus faecium, is commonly used as a livestock probioticand, thus, could be utilized as a co-administration agent. Furthermore,it is also intended that any of the acid-producing species of probioticor nutritional bacteria known in the art can be used in the compositionscomprising a microbial consortium as described herein.

A nutrient supplement comprising the microbial consortium as describedherein can include any of a variety of nutritional agents, includingvitamins, minerals, essential and nonessential amino acids,carbohydrates, lipids, foodstuffs, dietary supplements, short chainfatty acids and the like. Preferred compositions comprise vitaminsand/or minerals in any combination. Vitamins for use in a composition asdescribed herein can include vitamins B, C, D, E, folic acid, K, niacin,and like vitamins. The composition can contain any or a variety ofvitamins as may be deemed useful for a particularly application, andtherefore, the vitamin content is not to be construed as limiting.Typical vitamins are those, for example, recommended for dailyconsumption and in the recommended daily amount (RDA), although preciseamounts can vary. The composition can preferably include a complex ofthe RDA vitamins, minerals and trace minerals as well as those nutrientsthat have no established RDA, but have a beneficial role in healthyhuman or mammal physiology. The preferred mineral format would includethose that are in either the gluconate or citrate form because theseforms are more readily metabolized by lactic acid bacteria. In a relatedembodiment, the compositions described herein are contemplated tocomprise a microbial consortium in combination with a viable lactic acidbacteria in combination with any material to be adsorbed, including butnot limited to nutrient supplements, foodstuffs, vitamins, minerals,medicines, therapeutic compositions, antibiotics, hormones, steroids,and the like compounds where it is desirable to insure efficient andhealthy absorption of materials from the gastrointestinal tract into theblood. The amount of material included in the composition can varywidely depending upon the material and the intended purpose for itsabsorption, such that the composition is not to be considered aslimiting.

In some embodiments, the compositions described herein can furtherinclude a prebiotic and/or a fiber. Many forms of “fiber” exhibit somelevel of prebiotic effect. Thus, there is considerable overlap betweensubstances that can be classified as “prebiotics” and those that can beclassified as “fibers”. Non-limiting examples of prebiotics suitable foruse in the compositions and methods include psyllium,fructo-oligosaccharides, inulin, oligofructose,galacto-oligosaccharides, isomalto-oligosaccharidesxylo-oligosaccharides, soy-oligosaccharides, gluco-oligosaccharides,mannan-oligosaccharides, arabinogalactan, arabinxylan, lacto sucrose,gluconannan, lactulose, polydextrose, oligodextran,gentioligosaccharide, pectic oligosaccharide, xanthan gum, gum arabic,hemicellulose, resistant starch and its derivatives, and mixtures and/orcombinations thereof. The compositions can comprise from about 100 mg toabout 100 g, alternatively from about 500 mg to about 50 g, andalternatively from about 1 g to about 40 g, of prebiotic, per day or ona less than daily schedule.

Aspects of the technology described herein also include short chainfatty acids (SCFAs) and medium chain triglycerides (MCTs). Short chainfatty acids can have immunomodulatory (i.e., immunosuppressive) effectsand therefore their production (i.e., biosynthesis or conversion byfermentation) is advantageous for the prevention, control, mitigation,and treatment of autoimmune and/or inflammatory disorders(Lara-Villoslada F. et al., 2006. Eur J Nutr. 45(7): 418-425). Ingerm-free mice and vancomycin-treated conventional mice, administrationof SCFA (acetate, propionate, or butyrate) restored normal numbers ofTregs in the large intestine (Smith P M, et al. Science. 2013; 569-573).Short-chain fatty acids (SCFA) are produced by some bacteria as abyproduct of xylose fermentation. SCFA are one of the most abundantmetabolites produced by the gut microbiome, particularly the familyClostridiaceae, including members of the genus Clostridium,Ruminococcus, or Blautia. In some aspects, the pharmaceuticalcomposition, dosage form, or kit comprises at least one type of microbe(e.g., one or more microbial species, such as a bacterial species, ormore than one strain of a particular microbial species) and at least onetype of prebiotic such that the composition, dosage form, or kit iscapable of increasing the level of one or more immunomodulatory SCFA(e.g., acetate, propionate, butyrate, or valerate) in a mammaliansubject. Optionally, the pharmaceutical composition, dosage form, or kitfurther comprises one or more substrates of one or more SCFA-producingfermentation and/or biosynthesis pathways. In certain embodiments, theadministration of the composition, dosage form, or kit to a mammaliansubject results in the increase of one or more SCFAs in the mammaliansubject by approximately 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold,50-fold, 100-fold, or greater than 100-fold. In some embodiments, thedysbiosis is caused by a deficiency in microbes that produce short chainfatty acids. Accordingly, in some embodiments, the probiotic compositioncan contain a species of bacteria that produce short chain fatty acids.

MCTs passively diffuse from the GI tract to the portal system (longerfatty acids are absorbed into the lymphatic system) without requirementfor modification like long-chain fatty acids or very-long-chain fattyacids. In addition, MCTs do not require bile salts for digestion.Patients who have malnutrition or malabsorption syndromes are treatedwith MCTs because they do not require energy for absorption, use, orstorage. Medium-chain triglycerides are generally considered a goodbiologically inert source of energy that the human body finds reasonablyeasy to metabolize. They have potentially beneficial attributes inprotein metabolism, but may be contraindicated in some situations due totheir tendency to induce ketogenesis and metabolic acidosis. Due totheir ability to be absorbed rapidly by the body, medium-chaintriglycerides have found use in the treatment of a variety ofmalabsorption ailments. MCT supplementation with a low-fat diet has beendescribed as the cornerstone of treatment for primary intestinallymphangiectasia (Waldmann's disease). MCTs are an ingredient inparenteral nutritional emulsions.

Also contemplated herein are kits comprising, at a minimum, a microbialconsortium prep or formulations comprising all of the members of theconsortium in an admixture or comprising all of the members of theconsortium in sub-combinations or sub-mixtures. In some embodiments, thekit further comprises empty capsules to be filled by the practitionerand/or one or more reagents for enteric coating such capsules. It isalso contemplated herein that the microbe preparation is provided in adried, lyophilized or powdered form. In one embodiment, the kitscomprise at least 4 strains selected from the group consisting of:Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis,Clostridium bifermentans, Clostridium leptum, Clostridium sardiniensis,Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus,Parabacteroides goldsteinii, Prevotella tannerae, Clostridum hathewayi,Clostridum nexile, Clostridium hylemonae, Clostridiumglycyrrhizinilyticum, Clostridium scindens, Clostridium lavalense,Clostridum fimetarium, Clostridium symbiosum, Clostridiumsporosphaeroides, Dialister proprionicfaceins, Dialistersuccinatiphilus, Parabacteroides distasonis, Parabacteroidesgoldsteinii, Parabacteroides merdae, Peptostreptococcus anaerobius,Subdoligranulum variabile, and Veilonella ratti. In another embodiment,the kits comprise at least four strains selected from the groupconsisting of: Clostridium ramosum, C. scindens, C. hiranonsis, C.bifermentans, C. leptum, C. sardiniensis, Bacteroides fragilis, B.thetaiotaomicron, B. ovatus, Parabacteroides goldsteinii, and Prevotellatannerae. In another embodiment, the kits comprise at least four strainsselected from the group consisting of: Clostridium ramosum, C. scindens,C. hiranonsis, C. bifermentans, C. leptum, and C. sardiniensis. Inanother embodiment, the kits comprise at least four strains selectedfrom the group consisting of: Bacteroides fragilis, B. thetaiotaomicron,B. ovatus, Parabacteroides goldsteinii, and Prevotella tannerae. In oneembodiment, the kits comprise Bacteroides thetaiotaomicron orBacteroides fragilis and at least one of a strain selected from thegroup consisting of: Clostridium ramosum, Clostridium scindens,Clostridium hiranonsis, Clostridium bifermentans, Clostridium leptum,Clostridium sardiniensis, Bacteroides ovatus, Parabacteroidesgoldsteinii, Prevotella tannerae, Clostridum hathewayi, Clostridumnexile, Clostridium hylemonae, Clostridium glycyrrhizinilyticum,Clostridium scindens, Clostridium lavalense, Clostridum fimetarium,Clostridium symbiosum, Clostridium sporosphaeroides, Dialisterproprionicfaceins, Dialister succinatiphilus, Parabacteroidesdistasonis, Parabacteroides goldsteinii, Parabacteroides merdae,Peptostreptococcus anaerobius, Subdoligranulum variabile, and Veilonellaratti. In one embodiment, the kits comprise Bacteroides thetaiotaomicronor Bacteroides fragilis and at least one of a strain selected from thegroup consisting of: Clostridium ramosum, Clostridium scindens,Clostridium hiranonsis, Clostridium bifermentans, Clostridium leptum,Clostridium sardiniensis, Clostridum hathewayi, Clostridum nexile,Clostridium hylemonae, Clostridium glycyrrhizinilyticum, Clostridiumscindens, Clostridium lavalense, Clostridum fimetarium, Clostridiumsymbiosum, and Clostridium sporosphaeroides. In another embodiment, thekit comprises at least one reducing agent such as N-acetylcysteine,cysteine, or methylene blue for growing, maintaining and/orencapsulating the microbes under anaerobic conditions. The kitsdescribed herein are also contemplated to include cell growth media andsupplements necessary for expanding the microbial preparation. The kitsdescribed herein are also contemplated to include one or more prebioticsas described herein.

Prior to administration of the bacterial composition, the patient mayoptionally have a pretreatment protocol to prepare the gastrointestinaltract to receive the bacterial composition. In certain embodiments, thepretreatment protocol is advisable, such as when a patient has an acuteinfection with a highly resilient pathogen. In other embodiments, thepretreatment protocol is entirely optional, such as when the pathogencausing the infection is not resilient, or the patient has had an acuteinfection that has been successfully treated but where the physician isconcerned that the infection may recur. In these instances, thepretreatment protocol can enhance the ability of the bacterialcomposition to affect the patient's microbiome. In an alternativeembodiment, the subject is not pre-treated with an antibiotic.

As one way of preparing the patient for administration of the microbialecosystem, at least one antibiotic can be administered to alter thebacteria in the patient. As another way of preparing the patient foradministration of the microbial ecosystem, a standard colon-cleansingpreparation can be administered to the patient to substantially emptythe contents of the colon, such as used to prepare a patient for acolonoscopy. By “substantially emptying the contents of the colon,” thisapplication means removing at least 75%, at least 80%, at least 90%, atleast 95%, or about 100% of the contents of the ordinary volume of coloncontents. Antibiotic treatment can precede the colon-cleansing protocol.

If a patient has received an antibiotic for treatment of an infection,or if a patient has received an antibiotic as part of a specificpretreatment protocol, in one embodiment the antibiotic should bestopped in sufficient time to allow the antibiotic to be substantiallyreduced in concentration in the gut before the bacterial composition isadministered. In one embodiment, the antibiotic may be discontinued 1,2, or 3 days before the administration of the bacterial composition. Inone embodiment, the antibiotic can be discontinued 3, 4, 5, 6, or 7antibiotic half-lives before administration of the bacterialcomposition. If the pretreatment protocol is part of treatment of anacute infection, the antibiotic may be chosen so that the infection issensitive to the antibiotic, but the constituents in the bacterialcomposition are not sensitive to the antibiotic.

Any of the preparations described herein can be administered once on asingle occasion or on multiple occasions, such as once a day for severaldays or more than once a day on the day of administration (includingtwice daily, three times daily, or up to five times daily). Or thepreparation can be administered intermittently according to a setschedule, e.g., once weekly, once monthly, or when the patient relapsesfrom the primary illness. In another embodiment, the preparation can beadministered on a long-term basis to assure the maintenance of aprotective or therapeutic effect.

In one embodiment, a first microbial consortium comprising at least onebacterial species known to enhance colonization of beneficial organisms(e.g., Bacteroides thetaiotaomicron) is administered to a subject priorto administration of a second microbial consortium.

Another aspect described herein relates to a method for enhancing thecolonization and/or persistence of a microbial consortium, the methodcomprising administering a first microbial consortium comprising atleast 4 bacterial strains selected from the group consisting of:Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, Parabacteroidesgoldsteinii, and Prevotella tannerae, to a subject prior toadministering a second microbial consortium comprising at least 4bacterial strains selected from the group consisting of: Clostridiumramosum, C. scindens, C. hiranonsis, C. bifermentans, C. leptum and C.sardiniensis, wherein the first microbial consortium enhances thecolonization and/or persistence of the second microbial consortium.

It is also contemplated herein that a first microbial consortiumcomprising at least 4 bacterial strains selected from the groupconsisting of: Clostridium ramosum, C. scindens, C. hiranonsis, C.bifermentans, C. leptum and C. sardiniensis, is administered to asubject prior to administering a second microbial consortium comprisingat least 4 bacterial strains selected from the group consisting of:Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, Parabacteroidesgoldsteinii, and Prevotella tannerae.

It is also contemplated herein that a first microbial consortiumcomprising at least 4 bacterial strains selected from the groupconsisting of: Clostridium ramosum, C. scindens, C. hiranonsis, C.bifermentans, C. leptum and C. sardiniensis, is administered to asubject in combination with (e.g., simultaneously) a second microbialconsortium comprising at least 4 bacterial strains selected from thegroup consisting of: Bacteroides fragilis, B. thetaiotaomicron, B.ovatus, Parabacteroides goldsteinii, and Prevotella tannerae.

Efficacy

Typically, a food allergy response can manifest with one of more of thefollowing symptoms or indicators: (i) a marked drop in core bodytemperature, (ii) an increase in total IgE, (iii) an increase inallergen-specific IgE, (iv) mast cell expansion, (v) release of mastcell granule protease 1 (MMCP-1) and (vi) increase in Th2 cell skewing.Thus, efficacious treatment and/or prevention of food allergy using themethods and compositions described herein can reduce or eliminate atleast one of the symptoms or indicators associated with food allergy, asdescribed above. “Reduced” symptoms or indicators mean at least 20%reduced, at least 30% reduced, at least 40% reduced, at least 50%reduced, at least 60% reduced, at least 70% reduced, at least 80%reduced, at least 90% reduced, at least 95% reduced, at least 98%reduced or even at least 99% or further reduction. Methods for themeasurement of each of these parameters are known to those of ordinaryskill in the art.

The methods and compositions described herein provide treatment orprevention of food allergy involving or provoking anaphylaxis—i.e.,IgE-mediated histamine release or direct allergen-mediated degranulationof mast cells and basophils and resulting pathology. Non-limitingexamples include allergy or anaphylactic reaction to peanut, tree nuts,and shellfish, among others noted elsewhere herein. Food sensitivity,e.g., lactose intolerance or gluten intolerance involves differentmechanisms. While it is contemplated that a microbial consortium asdescribed herein can benefit those with food sensitivities (e.g., byreducing or eliminating a dysbiotic state and thereby reducing gutinflammation), the distinction between food sensitivities and foodallergies should be specifically noted. First and foremost,sensitivities do not provoke an anaphylactic response.

Effective prevention of food allergy can be assessed using an acceptedanimal model, such as that described herein or others known to those ofordinary skill in the art, wherein a regimen that sensitizes the animalsto a given food allergen in the absence of microbial consortiumtreatment fails to provoke a substantial allergic response in animalsadministered a protective microbial consortium as described herein. Asused herein, the term “fails to provoke a substantial allergic response”means that there is less than 20% of the allergic response (as measuredby one or more of the criteria (i)-(vi) described above) seen in animalssensitized to the allergen but without administration of a protective ortherapeutic microbial consortium as described herein. In human clinicalpractice, prevention or cure can be evaluated by administration of thegiven microbial consortium followed by administration of an allergenunder controlled circumstances in a doctor's office or hospital setting.For prevention, the microbial consortium can be administered prior to apatient's initial exposure to or consumption of a given food allergen.For therapy for established food allergy, the microbial consortium canbe administered as described herein, followed by consumption of the foodallergen in a controlled clinical setting. A lack of allergic reaction,or even a reduced allergic reaction relative to the patient's previousallergic responses to the allergen (i.e., at least 20% reduced, at least30% reduced, at least 40% reduced, at least 50% reduced, at least 60%reduced, at least 70% reduced, at least 80% reduced, at least 90%reduced, at least 95% reduced, at least 98% reduced or even at least 99%or further reduction) is evidence of effective treatment.

Repeated administration of the microbial consortium may be beneficial tomaintain a protective or curative effect.

In addition, efficacy of a particular formulation can be determined invitro or in an in vivo or in situ mouse model as described in herein oras known in the art (e.g., Noval Rivas et al. J Allergy Clin Immunol(2013) 131(1):201-212 or Noval Rivas et al., Immunity (2015) 42:512-523,the contents of which are each incorporated herein in their entirety).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

EXAMPLES

The data provided herein, e.g., in the figures and elsewhere, show thata microbial consortium of several species (i.e., 2, 3, 4, 5, or 6species, for example) can protect against developing food allergy in amouse model. Treatment with such a consortium of bacteria can reverseTH2 programming of Tregs. Treatment and/or prevention of food allergyusing a similar microbial consortium of microbes in humans isspecifically indicated.

Further methods for testing or measuring the efficacy of a microbialconsortium in a mouse model of food allergy are known in the art and/orcan be found in e.g., Noval Rivas et al. J Allergy Clin Immunol (2013)131(1):201-212 or Noval Rivas et al., Immunity (2015) 42:512-523, thecontents of which are each incorporated herein in their entirety.

Example 1: Therapeutic Microbiota to Treat Food Allergy Summary

Food allergy is a growing national problem, affecting 6% of children,and 3% of US teens and adults. Unfortunately for these children andtheir families, the standard of care remains to avoid offending foodsand manage symptoms as they occur. Therapies using oral desensitization,alone or with anti-IgE (Omalizumab™), remain experimental with limitedsuccess. Needed are therapies that target the aberrant immune responses.As such, this study shows the use of gut microbiota as a therapeuticintervention to promote tolerizing responses that can prevent ormitigate effects of Th2/allergic responses.

Food allergies occur with development of Th2-allergic responses tofoodstuffs, in contrast to tolerizing T-regulatory responses thatmitigate such responses mucosally. The Th2 responses promote foodantigen-specific IgE antibody and recruitment of mucosal mast cells, incontrast to regulatory responses, which inhibit these effects. Oncesensitized to one or more food antigens, re-exposure can inducelife-threatening anaphylactic responses. Capacity to promote tolerizingresponses supports a broad-based therapeutic approach that can act atthe earliest stages of exposure as well as in the already-sensitizedpatient to prevent aberrant allergic responses across a spectrum offoodstuffs.

Leveraging the genetically susceptible IL4RA F709 mouse model [1,2] offood allergy defined human commensal communities that can both preventand cure food allergies in preclinical models have been developed. Thesecommunities leverage a new therapeutic pathway forpatients—immunomodulation from the luminal side of the gut, the space inwhich the gut microbiota resides. Human gut microbiota consists of manyhundreds of species that provide critical functions in normal humandevelopment and health, from maturing of the immune system, providingessential nutrients such as B vitamins and vitamin K, and assisting indigestion and metabolism of dietary and exogenous compounds, includingdrugs and ingested foodstuffs.

Background

The therapeutic consortia consist of defined and culturable humancommensal strains that stimulate protective regulatory T cell responsesin the gut. Two protective communities have been developed, Gut-ProtectI (GP-I) and Gut-Protect II (GP-II). All have complete or contig-levelgenomic sequences, and select members from each group can be geneticallymanipulated. In addition, a third community has been developed thatdemonstrates worsened effects (Negative Control Consortium, Neg-CC),which is providing further information on dysbiotic effects in vivo andpotential biomarkers to assist in diagnosis and selection of therapy.

(1) Gut-protect I: Community of culturable gut commensals that hasdemonstrated treat-to-protect and treat-to-cure efficacy in food antigensensitization of IL4RA F709 mice.

(2) Gut-protect II: Community of culturable gut commensals that hasdemonstrated robust treat-to-prevent efficacy in animal models and lessrobust treat-to-cure efficacy than Gut-protect I. However, in standardsolutions administered to the host, this community tolerates greaterambient air exposure than the Gut-protect I community.

(3) Negative Control Community: Community of culturable gut commensalsthat promote food antigen sensitization in treat-to-prevent andtreat-to-cure regimens. This community is defining the nature ofdysbiosis in food allergy and supporting development of biomarkers thatcould predict microbiota-derived factors that enhance susceptibility todevelopment of food allergies.

In pre-clinical models of food allergy to egg, leveraging a susceptiblemouse model that does not require the use of adjuvants, GP-I and GP-IIeach demonstrated treat-to-prevent efficacy, when used in a period ofsensitization to the allergy-inducing foodstuff. GP-I was also effectivein treat-to-cure regimens when used in animals that had already beensensitized to egg protein, to the point that re-exposure would inducelife-threatening anaphylactic responses. In contrast, the negativecontrol community worsened effects, and provides further insights as tothe underlying dysbiosis that may contribute to development of foodallergies, while also providing potential biomarkers to identifyindividuals at risk, or for whom microbial therapy may be efficacious.

Consortia Development

For pre-clinical studies in mice the component members are grownindividually in nutrient-rich media under appropriate anaerobicconditions, quantitated for biomass, and then the consortium is mixedunder anaerobic conditions with approximately equal biomass of eachcomponent organism to a final concentration ˜5.0×10⁸ colony formingunits (CFU)/mL. Input culture volumes for each species have been in therange of 100 mL-1 L. As needed, cultures with a stationary phase biomass<5×10⁸ CFU/mL are concentrated by centrifugation with re-suspensionhandled under anaerobic conditions.

When mixed, the total biomass remains approximately 5×10⁸ CFU/mL. 2 mLaliquots are placed in cryovials with an anaerobic/pre-reducedatmosphere, snap frozen on liquid nitrogen and stored at −80° C. untiluse. Rapid freezing has shown to have <½ log effects on the biomass ofthe component organisms and no effect on efficacy in animal models. Forstudies, tubes are thawed and mice administered 200 uL of this solutionweekly to twice weekly by oral gavage, resulting in a total introducedbiomass of 1×10⁸ CFU/mouse. The measurements of gut contents in adultmice (stomach through anus) range from 4-8 mL of material. The gavagedconsortium is thus 2.5-5% of the total volume of contents in the mousegut and >10% of the volume of contents in the small bowel.

In terms of pre-existing microbial biomass from the conventionalmicrobiota—the mouse small intestine on average has ˜10⁴ CFU/mL inproximal duodenum with increase to 10⁸ CFU/mL in the ileum. Biomassincreases to 10⁹-10¹⁰ CFU/mL in the cecum and colon. From the standpointof microbial biomass at locations in the mouse small bowel, the primarysite of action of the consortia to promote regulatory T cell responses,the consortium is 10,000× the biomass of the duodenal microbiota, and1-2× the biomass of the jejunal and ileal microbiota.

In comparison, the adult human gut may contain 4.5 L of material, ofwhich 1 L relates to ingested foodstuffs with 3.5 L of secretionsincluding saliva, bile, and other fluids from the pancreas andintestines [3]. These fluids and electrolytes are largely resorbed inthe right colon, subsequent to fecal compaction and passage. Within theintestines, the biomass of organisms also varies, with the highestconcentration in the cecum and right side of the colon (10¹⁰-10¹²CFU/mL). In contrast, in the small intestine—the believed site ofaction, the biomass also ranges from 10⁴ CFU/mL in the duodenum to 10⁸CFU/mL in the ileum [4, 5].

Human Dosing

The CFU/dose for humans is based on the following parameters:

(1) Treatment of C. difficile with oral capsule formulations of humanstool: Data from OpenBiome and other groups have shown successfultreatment of C. difficile colitis with capsule formulation thatadministers 3-5×10⁹ CFU in a range of 12-30 capsules taken per one-timedose [6]. A standard 12-capsule regimen is expected to deliverapproximately 4.2×10⁹ CFU per dose.

(2) Alter the small intestinal microbiota to promote immunomodulation.An encapsulated formulation releasing contents in the proximal smallbowel will deliver a dose of 3-5×10⁹ CFU, exceeding the duodenal biomassby a factor of 10,000, and approaching a 1:1 ratio with communities inthe jejunum and ileum.

Other formulations including nanoparticles in liquid, with optionalpre-biotic compounds to enhance colonization and viability, or areconstituted lyophilisate are contemplated, however given the need toprevent exposure to oxygen and for ease of storage and administration,the first formulation uses encapsulated material.

Administration

Given the obligately anaerobic nature of the component species, phase Istudies will use encapsulated formulations with the followingproperties:

Stage I:

-   -   Can be swallowed by an adult or child >8 years of age.    -   Excludes oxygen    -   Holds a volume so that a person needs to take 15 or less        capsules per dose    -   Can be stored frozen (−20° C. or −80° C.) and thawed prior to        administration    -   Releases contents after passage through the stomach

In one embodiment, the capsules used by OpenBiome™ for oral FMT therapyare used to encapsulate the GP-I and GP-II mixtures [i]. Other optionsare also available commercially and contemplated herein. In someembodiments, the capsules consist of frozen material (in order to ensurean adequate product) that is thawed prior to administration and isencapsulated, free of oxygen, with material that survives intact intothe small intestine.

Scale of Culture

The animal studies used pilot cultures in the range of 100-1000 mL. Togenerate human doses, the culture is scaled by at least a factor of 10.The following steps are contemplated herein.

-   -   (1) Perform growth curves in different media conditions—to        optimize growth conditions and correlate an OD600 with plated        biomass.    -   (2) Grow the component members anaerobically in liquid media.        Media is pre-reduced and incubated at 37° C. with some level of        agitation (e.g., 150 rpm or with stirring/fermenter baffles) to        insure a maximal culture density. Depending upon the fermenter        system, nitrogen or anaerobic gas mixtures can be sparged to        maintain anaerobic conditions. However, none of the component        species require H₂ or CO₂ for growth, beyond maintaining        appropriate acid/base balance.    -   (3) Concentrate select members as needed to obtain desired input        density: commonly done by centrifugation at 5-10K RPM with        pull-off of supernatant under anaerobic conditions and        resuspension in a lesser volume of new culture media or        appropriate suspension buffer. The new culture density is        confirmed by OD600 reading and viability by plating to solid        media.    -   (4) Aggregate the cultured into the combined consortium:        Estimated biomass from the OD600 readings are used to estimate        the volume and prepare the aggregate.    -   (5) Prepare capsules: done under anaerobic conditions to        preserve viability.    -   (6) Store capsules: optimal to store at conditions available        clinically, e.g. −20° C.    -   (7) Quality Control: In addition to QC for prior steps and        media, the final community will be evaluated to insure the        appropriate species are present and in desired viable biomass.        Analyses on materials for pre-clinical studies used 16S rRNA        gene phylotyping with culture a qPCR-based methods. Metagenomic        approaches may also be used to rule-out contamination with        nonbacterial species or viruses.

It is further contemplated herein that growth conditions are optimizedfor the scaled cultures.

In some embodiments, media formulations are developed such that theylack animal products and/or substrates that might be associated withsensitizing antigens in foodstuffs. In addition, one of skill in the artcan assess if additives to the inoculum enhance viability in capsulesand once released in vivo. Materials can include preservatives andprebiotic compounds.

Stage II:

It is further contemplated herein that the consortia described hereinare formulated as a liquid formulation that can be administered toinfants and young children. It is contemplated herein that suchformulations comprise mixtures of spores from sporulating species,leveraging non-sporulating obligate anaerobes with limitedaerotolerance, and including reducing factors in a liquid formula tobuffer against short-term exposure to oxygen in ambient air and uponentry into the digestive tract. Compounds such as the amino acidcysteine or n-acetylcysteine, which have been used therapeutically ininfants and have a robust safety profile are contemplated [7,8].

Additional pre-clinical animal models for use in testing formulationsinclude e.g., neonatal swine models of food allergy, including ones forfoodstuffs common in the diet of both humans and pigs [9].

REFERENCES

-   1. Mathias C B, Hobson S A, Garcia-Lloret M, Lawson G, Poddighe D,    Freyschmidt E J, Xing W, Gurish M F, Chatila T A, Oettgen H C.    IgE-mediated systemic anaphylaxis and impaired tolerance to food    antigens in mice with enhanced IL-4 receptor signaling. J Allergy    Clin Immunol. 2011 March; 127(3):795-805.e 1-6.-   2. Noval Rivas M, Burton O T, Wise P, Zhang Y Q, Hobson S A, Garcia    Lloret M, Chehoud C, Kuczynski J, DeSantis T, Warrington J, Hyde E    R, Petrosino J F, Gerber G K, Bry L, Oettgen H C, Mazmanian S K,    Chatila T A. A microbiota signature associated with experimental    food allergy promotes allergic sensitization and anaphylaxis. J    Allergy Clin Immunol. 2013 January; 131(1):201-12.-   3. Secretions in the GI tract—available on the world wide web at:    en.wikibooks.org/wiki/Medical_Physiology/Gastrointestinal_Physiology/Secretions-   4. Genes Nutr. 2011 August; 6(3): 209-240-   5. Stool substitute transplant therapy for the eradication of    Clostridium difficile infection: ‘RePOOPulating’ the gut. Microbiome    Journal Open Access (2013) DOI: 10.1186/2049-2618-1-3.-   6. Open Biome FMTcapsules F30 and G3: available on the world wide    web at openbiome.org/fmtcapsules/-   7. Zlotkin, S H, et al. Cysteine supplementation to cysteine-free    intravenous feeding regimens in newborn infants. Am J Clin Nutr.    1981, 34(5): 914-923.-   8. Marzullo, L. An update of N-acetylcysteine treatment for acute    acetaminophen toxicity in children. Curr Op Peds. 2005, 17(2):    239-245.-   9. Helm, R H et al. A neonatal swine model for peanut allergy. JACI.    2002, 109(1): 136-142.

Example 2: OTU Clustering Method for Data from Human and Animal Studies

DNA Extraction and Sequencing for 16S rRNA Gene Phylotyping.

A multiplexed amplicon library covering the V4 region of the 16S rDNAgene was generated from DNA extracted from human stool, mouse fecalpellets or segments of snap-frozen gut tissues using MO BIO™Power-Fecal™ DNA Isolation Kits (MO BIO™ Laboratories) with custommodification to enhance lysis of Gram positive commensals with thickcell walls. The rest of library preparation followed the protocol of [1]with dual-index barcodes. Aggregated libraries are sequenced withpaired-end 250 bp reads on the Illumina™ MiSeq platform. The aggregatelibrary pool was size selected from 300-500 bp on a Pippin™ prep 1.5%agarose cassette (Sage Sciences™) according to the manufacturer'sinstructions. Concentration of the pool is measured by qPCR (KapaBiosystems™) and loaded onto the MiSeg™ (Illumina™) at 6-9 pM with 20%phiX spike-in to compensate for low base diversity according toIllumina™'s standard loading protocol.

16S rRNA Data Preprocessing.

Sequencing aims to obtain 10-50K usable reads per sample after qualityfiltering. Raw sequencing reads were processed using the mothur softwarepackage (v.1.35.1)[2] and custom Python and R scripts [3], which performde-noising, quality filtering, alignment against the ARB Silva referencedatabase of 16S rDNA gene sequences, and clustering into OperationalTaxonomic Units (OTUs) at 97% identity.

16S rRNA Data Analysis.

To statistically test for differences between control and food allergicsubjects in abundances of microbial taxa (OTUs), the DESeq2 softwarepackage was employed to support of analyses relative to host co-variatessuch as age, food allergy status, diet and antibiotic use in humancohort, OTUs showing significant differences were defined by: (1)adjusted p-value <=0.1; (2) relative abundance >=0.01 in either controlor food allergic groups; (3) absolute value of log 2 fold changes >=2.

To improve the resolution of taxonomic calls and show phylogeneticrelationships, a separate method used the pplacer software package toperform phylogenetic placement of individual OTU [4]. Pplacer uses alikelihood-based methodology to place short sequencing reads of 16S rRNAamplicons on a reference tree, and also generates taxonomicclassifications of the short sequencing reads using a least commonancestor-based algorithm. The reference tree required for phylogeneticplacement is generated using full-length or near full-length (>1,200 nt)16S rDNA sequences of type strains from the Ribosomal Database Project(RDP)[5].

For all statistical testing for 16S rDNA data analysis, p-values wereadjusted for multiple hypothesis testing using the method of Benjaminiand Hochberg (BH) [6]. Heat map plots are generated using custom Rscripts [3].

Alpha diversity values (richness of a sample in terms of the diversityof the OTUs observed in it) were calculated using Shannon entropy tomeasure diversity in each sample. Beta-diversity values (distancebetween samples based on differences in OTUs present in each sample)were calculated using the unweighted/weighted Unifrac dissimilaritymeasure, to assess differences in overall microbial community structure.

(3) OTU Mappings of the Defined Species

The following operational taxonomic units map to the defined species, asidentified in gnotobiotic mice colonized with these consortia. Fecalpellets were subjected to the above described 16S rRNA gene phylotypingover the V4 variable region.

TABLE 2 Mapping of the defined therapeutic species to OTU based on the16S rRNA V4 region. Species OTU taxonomic mappings, V4 region-Bacteroides Bacteria: Bacteroidetes: Bacteroidia: Bacteroidales fragilisBacteria: Bacteroidetes: Bacteroidia: Bacteroidales: BacteroidaceaeBacteria: Bacteroidetes: Bacteroidia: Bacteroidales: Bacteroidaceae:Bacteroides Bacteroides Bacteria: Bacteroidetes: Bacteroidia:Bacteroidales thetaiotaomicron Bacteria: Bacteroidetes: Bacteroidia:Bacteroidales: Bacteroidaceae Bacteria: Bacteroidetes: Bacteroidia:Bacteroidales: Bacteroidaceae: Bacteroides Bacteroides Bacteria:Bacteroidetes: Bacteroidia: Bacteroidales ovatus Bacteria:Bacteroidetes: Bacteroidia: Bacteroidales: Bacteroidaceae Bacteria:Bacteroidetes: Bacteroidia: Bacteroidales: Bacteroidaceae: BacteroidesClostridium Bacteria: Firmicutes: Clostridia: Clostridiales:Peptostreptococcaceae bifermentans Bacteria: Firmicutes: Clostridia:Clostridiales: Peptostreptococcaceae: Clostridium cluster XI ClostridiumBacteria: Firmicutes: Clostridia: Clostridiales: Peptostreptococcaceaehiranonsis Bacteria: Firmicutes: Clostridia: Clostridiales:Peptostreptococcaceae: Clostridium cluster XI Clostridium Bacteria:Firmicutes: Clostridia: Clostridiales: Ruminococcaceae leptum Bacteria:Firmicutes: Clostridia: Clostridiales: Ruminococcaceae: Clostridiumcluster IV Clostridium Bacteria: Firmicutes: Erysipelotrichia:Erysipelotrichales: Erysipelotrichaceae ramosum Erysipelotrichales:Erysipelotrichaceae: Clostridium cluster XVIII Clostridium Bacteria:Firmicutes: Clostridia: Clostridiales: sardiniensis Bacteria:Firmicutes: Clostridia: Clostridiales: Clostridiaceae I (absonum)Clostridium Bacteria: Firmicutes: Clostridia: Clostridiales:Lachnospiraceae scindens Bacteria: Firmicutes: Clostridia:Clostridiales: Lachnospiraceae: Clostridium cluster XIVa ParabacteroidesBacteria: Bacteroidetes: Bacteroidia: Bacteroidales: Porphyoromondaceaegoldsteinii Bacteria: Bacteroidetes: Bacteroidia: Bacteroidales:Porphyoromondaceae: Parabacteroides Prevotella Bacteria: Bacteroidetes:Bacteroidia: Bacteroidales: tannerae Bacteria: Bacteroidetes:Bacteroidia: Bacteroidales: Prevotellaceae Bacteria: Bacteroidetes:Bacteroidia: Bacteroidales: Prevotellaceae: Prevotella

TABLE 3 Mapping of the dysbiotic consortium species to OTU based on the16S rRNA V4 region Species OTU mapping Bilophila Bacteria:Proteobacteria: Deltaproteobacteria: Desulfovibrionales wadsworthiaBacteria: Proteobacteria: Deltaproteobacteria: Desulfovibrionales:Desolfovibrionaceae Bacteria: Proteobacteria: Deltaproteobacteria:Desulfovibrionales: Desolfovibrionaceae: Bilophila EnterobacterBacteria: Proteobacteria: Gammaproteobacteria: Enterobacteriales cloacaeBacteria: Proteobacteria: Gammaproteobacteria: Enterobacteriales:Enterobacteriaceae Bacteria: Proteobacteria: Gammaproteobacteria:Enterobacteriales: Enterobacteriaceae: Enterbacter Escherichia Bacteria:Proteobacteria: Gammaproteobacteria: Enterobacteriales coli Bacteria:Proteobacteria: Gammaproteobacteria: Enterobacteriales:Enterobacteriaceae Bacteria: Proteobacteria: Gammaproteobacteria:Enterobacteriales: Enterobacteriaceae: Escherichia Klebsiella Bacteria:Proteobacteria: Gammaproteobacteria: Enterobacteriales pneumoniaBacteria: Proteobacteria: Gammaproteobacteria: Enterobacteriales:Enterobacteriaceae Bacteria: Proteobacteria: Gammaproteobacteria:Enterobacteriales: Enterobacteriaceae: Klebsiella Proteus Bacteria:Proteobacteria: Gammaproteobacteria: Enterobacteriales mirabilisBacteria: Proteobacteria: Gammaproteobacteria: Enterobacteriales:Enterobacteriaceae Bacteria: Proteobacteria: Gammaproteobacteria:Enterobacteriales: Enterobacteriaceae: Proteus

TABLE 4 Additional “beneficial” OTU identified in the longitudinalpediatric human cohort as associated with protection from development offood allergy. Nearest species mapping(s) with OTU taxonomic mappingswith sequencing of the V4 region- pplacer Bacteria: Firmicutes:Clostridia: Clostridiales: Lachnospiraceae Clostridium hathewayiBacteria: Firmicutes: Clostridia: Clostridiales: Lachnospiraceae:Clostridium cluster XIVa Bacteria: Firmicutes: Clostridia:Clostridiales: Lachnospiraceae: Hungatella Bacteria: Firmicutes:Clostridia: Clostridiales: Lachnospiraceae Clostridium nexile, Bacteria:Firmicutes: Clostridia: Clostridiales: Lachnospiraceae: Clostridiumcluster Clostridium XIVa hylemonae, Clostridium glycyrrhizinilyticum,Clostridium scindens, Clostridium lavalense, Clostridium fimetarium,Clostridium symbiosum Bacteria: Firmicutes: Clostridia: Clostridiales:Ruminococcaceae Clostridium Bacteria: Firmicutes: Clostridia:Clostridiales: Ruminococcaceae: Clostridium sporosphaeroides cluster IVBacteria: Firmicutes: Negativicutes: Selenomonadales: VeillonellaceaeDialister Bacteria: Firmicutes: Negativicutes: Selenomonadales:Veillonellaceae: Dialister proprionicifaciens, Dialister succinatiphilusBacteria: Bacteroidetes: Bacteroidia: Bacteroidales: PorphyoromondaceaeParabacteroides Bacteria: Bacteroidetes: Bacteroidia: Bacteroidales:Porphyoromondaceae: distasonis, Parabacteroides Parabacteroidesgoldsteinii, Parabacteroides merdae Bacteria: Firmicutes: Clostridia:Clostridiales: Peptostreptococcaceae Peptostreptococcus Bacteria:Firmicutes: Clostridia: Clostridiales: Peptostreptococcaceae: anaerobiusPeptostreptococcus Bacteria: Firmicutes: Clostridia: Clostridiales:Ruminococcaceae Subdoligranulum Bacteria: Firmicutes: Clostridia:Clostridiales: Ruminococcaceae: Subdoligranulum variabile Bacteria:Firmicutes: Negativicutes: Selenomonadales: Veillonellaceae Veilonellaratti Bacteria: Firmicutes: Negativicutes: Selenomonadales:Veillonellaceae: Veillonella

TABLE 5 Additional “dysbiotic” OTU identified in the longitudinalpediatric human cohort as associated with development of food allergy.Nearest species mapping OTU taxonomic mappings with sequencing of the V4region with pplacer Bacteroidetes: Bacteroidia: Bacteroidales:Rikenellaceae: Alistipes Bacteroidetes: Bacteroidia: Bacteroidales:Rikenellaceae: Alistipes onderdonkii Firmicutes: Clostridia:Clostridiales: Lachnospiraceae Blautia Firmicutes: Clostridia:Clostridiales: Lachnospiraceae: Blautia wexlerae, Blautia henselaeBacteria: Proteobacteria: Deltaproteobacteria: DesulfovibrionalesBilophila Bacteria: Proteobacteria: Deltaproteobacteria:Desulfovibrionales: Desolfovibrionaceae wadsworthia, Bacteria:Proteobacteria: Deltaproteobacteria: Desulfovibrionales:Desolfovibrionaceae: Desulfovibrio Bilophila species Bacteria:Proteobacteria: Deltaproteobacteria: Desulfovibrionales:Desolfovibrionaceae: Bilophila: Desulfovibrio Firmicutes: Bacilli:Lactobacillales: Lactobacillaceae: Lactobacillus Lactobacillus johnsoniBacteria: Proteobacteria: Betaproteobacteria: Burkholderales:Parasutterella Bacteria: Proteobacteria: Betaproteobacteria:Burkholderales: Sutterellaceae excrementihominis Bacteria:Proteobacteria: Betaproteobacteria: Burkholderales: Sutterellaceae:Parasutterella Firmicutes: Clostridia: Clostridiales: LachnospiraceaeRoseburia Firmicutes: Clostridia: Clostridiales: Lachnospiraceae:Roseburia inulinivorans

REFERENCES

-   1. Kozich, J. J., et al., Development of a dual-index sequencing    strategy and curation pipeline for analyzing amplicon sequence data    on the MiSeq Illumina sequencing platform. Applied and environmental    microbiology, 2013. 79(17): p. 5112-5120.-   2. Schloss, P. D., et al., Introducing mothur: open-source,    platform-independent, community-supported software for describing    and comparing microbial communities. Appl Environ Microbiol, 2009.    75(23): p. 7537-41.-   3. McMurdie, P. and S. Holmes, phyloseq: An R package for    reproducible interactive analysis and graphics of microbiome census    data. PloS ONE, 2013. 8(4): p. e61217.-   4. Matsen, F., R. B. Kodner, and E. V. Armbrust, pplacer: linear    time maximum-likelihood and Bayesian phylogenetic placement of    sequences onto a fixed reference tree. BMC bioinformatics, 2010.    11(1): p. 538.-   5. Cole, J. R., et al., Ribosomal Database Project: data and tools    for high throughput rRNA analysis. Nucleic acids research, 2014.    42(D1): p. D633-42.-   6. Benjamini, Y. and Y. Hochberg, Controlling the false discovery    rate: a practical and powerful approach to multiple testing. Journal    of the Royal Statistical Society Series B, 1995. 57(1): p. 289-300.

Example 3: Microbiology-Level Activities Used in Selection of DefinedSpecies

The species in the defined consortia were selected per knownbiochemical, immunologic and microbiologic functions with capacity toaffect beneficial immunomodulatory responses in the host. Withoutwishing to be bound by theory, microbiologic mechanisms of action caninclude the following.

Adjuvant effects of microbial products to stimulate the development ofregulatory T cells through TLR->MyD88 and other immune cell pathways.The production of key microbial antigens from commensal anaerobes,including their lipoteichoic acid (LTA), exo-polysaccharides (PSA), LPS,bacterial flagellin, and bacterial DNA can act through stimulatingtoll-like-receptor pathways to skew mucosal T cells to a regulatory vs.allergic phenotype. In contrast, published data have shown thatbacterial cell wall fractions from members of the negative controlconsortium can promote aberrant stimulation of both allergic (Th2) andpro-inflammatory (Th1) responses. The distinct portions of thesemolecules that skew towards tolerance vs. allergy or inflammationhighlight the interplay between mammalian hosts and colonizingmicrobiota, including the microbial products that signal the host tomaintain a healthy homeostasis versus elicit pathogenic immuneresponses.

Mucosal and Immunoprotective Functions of Microbial End-Products ofMetabolism:

Short chain fatty acids (SCFA) are natural end-products of microbialanaerobic fermentation as are additional small molecule metabolites fromanaerobic fermentation of different carbon sources. End-products such asbutyrate have been shown to provide a primary energy source to the gutepithelium and to contribute to the development of tolerizing responsesin mucosal locations. The consortia selected produce a dominance ofbutyrate and propionate from the fermentation of simple and complexcarbohydrates that may be in the gut lumen, per the diet and secretionof host factors. These factors would likely act in combination withother microbial activities to mediate the desired immunomodulatoryeffects.

Biochemical Activities:

The species selected perform the full complement of bile acidtransformations and also transform a variety of other moleculesincluding other cholesterol-derivatives, biogenic amines, lipids andproduction of aryl hydrocarbons which may serve as microbialsiderophores, quorum sensing molecules and other metabolic intermediateswithin the microbial cell. Such metabolites are potentially capable ofstimulating host aryl-hydrocarbon receptor (AHR) pathways which havealso been demonstrated to promote tolerizing responses in the gutmucosa.

Gut Conditioning:

Microbiologically, select members of the consortia are known to aid thesubsequent colonization, biochemical and further immunoprotective rolesof other species. Both Bacteroides fragilis and Bacteroidesthetaiotaomicron, when included in defined flora, assist the growth ofmore fastidious members of the Bacteroidetes, Firmicutes andActinobacteria. Clostridium ramosum has demonstrated comparable effectsin defined colonizations of germ-free mice with other commensals.Effects are multi-factorial, and include maturing of gut epithelialresponses, altered host secretion of glycoconjugates which can serve ascarbon sources for the commensal flora, enhancing gut peristalsis anddigestion, reducing lumen gut oxygen tension so more obligatelyanaerobic species can flourish, and releasing metabolites, and/orextracellular products of microbial digestion which support the growthof additional species by providing carbon and/or nitrogen sources,vitamins, and other essential micronutrients.

Reducing the Biomass of Dysbiotic or Pathogenic Species:

Animal models conducted by our group have also shown that the species inthe gut protect communities can reduce the biomass of theProteobacterial species in the negative control consortium. Withoutwishing to be bound by theory, mechanistically these biomass reductionsalso reduce the antigen burden of products from these species thatpreferentially skew towards allergic responses.

Example 4. GP-I and GP-II Consortia are Effective in Treat-to-PreventFood Allergy in Conventional Wild-Type and IL4RA F709 Mice

The protective effects of consortia GP-I (Clostridial species) and GP-II(Bacteroidetes species) were examined in conventional and foodallergy-prone IL4RA F709 mice. In this series of experiments, theanimals were conventional, i.e., not germ-free. Animals were treatedwith a course of oral broad-spectrum antibiotics one week prior toinitiating sensitization with egg ovalbumin (OVA) and staphylococcalenterotoxin B (SEB). After antibiotic treatment, the mice receivedweekly doses of the GP-I, GP-II or negative control consortia (NCC), andweekly doses of OVA-SEB before challenge at week 8 with oral ovalbumin.See FIG. 21A. Following challenge, animals were examined for temperaturedrop (FIG. 21B), total and OVA-specific IgE titers (FIG. 21C), mast cellprotease-1 levels (FIG. 21D), mast cell recruitment to the small bowel(FIG. 2E) and the development of FoxP3+ regulatory T cells (FIGS. 21Fand G) versus IL-4 producing T cells (FIGS. 21H and I). The data clearlyshow that the Clostridiales and Bacteroidetes consortia (GP-I and GP-II,respectively) protected against allergy as measured by these criteria,while animals treated with the negative control consortium (denoted“Proteobacteria”) or no bacteria showed a clear allergic response. Thus,GP-1I and GP-II are effective to prevent the development of foodallergy.

Example 5. GP-I and GP-II Consortia Protect Germ-Free Mice inTreat-to-Prevent Regimens

The protective effects of the GP-I and GP-II consortia were examined ingerm-free mice treated with saline (PBS) or with OVA-SEB. The germ-freemice received either no bacteria or consortia GP-I or GP-II prior to 7weekly sensitizing doses of OVA-SEB (see FIG. 22A). Challenged on the8^(th) week with OVA, the mice were examined for evidence of allergicreaction as described in Example 4. The data (see FIGS. 22 B-H) clearlyshow that unlike animals receiving no bacteria, germ-free animalsadministered the GP-I and GP-II consortia experienced substantially noallergic reaction upon challenge with the sensitizing food allergen.

Example 6. GP-I and GP-II Consortia Cure Food Allergy in ConventionalIL4RA F709 Mice; Negative Control Consortium does not

To test whether animals that had been sensitized to food allergen beforetreatment with consortia shown herein to be protective against thedevelopment of food allergy could be cured of allergic sensitivity,conventional (i.e., non-germ-free) IL4RA F709 mice were sensitized toovalbumin by 8 weekly treatments with OVA-SEB as performed in theexperiments described above (see FIG. 23A for the experimentaltimeline). The animals were then treated with antibiotics to knock outtheir natural gut bacteria, before being treated with the GP-I, GP-II orNCC consortia using four weekly doses. After the fourth weekly dose ofthe test consortia, the animals were challenged with OVA. The data,which examined the same criteria examined in Examples 4 and 5, show thatanimals treated with the GP-I (Clostridiales) and GP-II (Bacteroidetes)consortia were substantially protected against allergic reaction, whilethose receiving the negative control (Proteobacteria) consortium werenot (see FIG. 23B-H). These data demonstrate that not only can foodallergy be prevented from developing through administration of themicrobial consortia described herein, but established food allergy canalso be treated by administration of the same protective consortia.

Example 7. GP-II Consortium (Bacteroidetes) Protects Against FoodAllergy without Prior Antibiotic Knockdown of the Flora

To examine whether it is necessary to first knock down the natural gutmicrobiota via antibiotic treatment in order to obtain protection inconventional mice, such animals were administered 8 weekly doses of GP-Iand GP-II consortia or no additional bacterial treatment, whilesensitizing mice to OVA with weekly OVA-SEB treatments over the courseof 8 weeks (see FIG. 24A for the experimental timeline). Subsequentchallenge with OVA demonstrated that, in the absence of natural floraknockdown prior to treatment with GP-I or GP-II, the Bacteroidetesconsortium, GP-II, provided protection against the development of foodallergy (see FIG. 24B-L). It is specifically contemplated that theadministration of a combination of GP-II with one or more, or even allof the species of GP-I would provide an improved effect, in terms ofinitial response or protection and/or in terms of the persistence ofsuch protection.

Example 8. The GP-I Consortium (Clostridiales) can Cure Food Allergywithout the Use of Antibiotics in Conventional Mice

To examine whether it is necessary to knock down the natural gutmicrobiota in order to cure food allergy in conventional mice,conventional wild-type and IL4RA F709 mutant mice were sensitized toovalbumin via 8 weekly doses of OVA-SEB, followed by twice-weeklyadministration of the GP-I (Clostridiales) consortium for 4 weeks,without prior antibiotic knockdown of the natural gut microbiota (seeFIG. 25A for the experimental timeline). When challenged after the fourweeks of GP-I administration, the data show that the GP-I consortium waseffective to reduce allergic symptoms by all measures examined in boththe wild-type and allergy-prone animals (See FIG. 25B-I). The dataindicate that the administration of the GP-I consortium is able to curefood allergy in the presence of the natural gut flora—that is, it is notnecessary to first knock down the natural gut microbiota to effectivelytreat food allergy using protective microbial consortia such as the GP-Iconsortium.

1. A pharmaceutical composition comprising: (i) a preparation comprising a minimal microbial consortium consisting essentially of four to eleven strains of viable gut bacteria, in an amount sufficient to increase immune system tolerance when administered to an individual in need thereof, and (ii) a pharmaceutically acceptable carrier.
 2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for oral delivery.
 3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is in the oral dosage form of a capsule, a reconstituted lyophilisate, a food item, or is formulated as a liquid, gel, or fluid-gel.
 4. The pharmaceutical composition of claim 1, wherein the four to eleven strains of viable gut bacteria consisting essentially of strains of Clostridium or Bacteroides.
 5. The pharmaceutical composition of claim 1, wherein the four to eleven strains of viable gut bacteria consisting essentially of strains of Clostridium.
 6. The pharmaceutical composition of claim 1, wherein the four to eleven strains of viable gut bacteria consisting essentially of strains of Bacteroides.
 7. The pharmaceutical composition of claim 1, wherein the four to eleven strains of viable gut bacteria comprise Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis, Clostridium bifermentans, Clostridium leptum, Clostridium sardiniensis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus, Parabacteroides goldsteinii, or Prevotella tannerae.
 8. The pharmaceutical composition of claim 1, wherein the four to eleven strains of viable gut bacteria comprise Clostridium ramosum, Clostridium scindens, Clostridium hiranonsis, Clostridium bifermentans, Clostridium leptum, or Clostridium sardiniensis.
 9. The pharmaceutical composition of claim 1, wherein the four to eleven strains of viable gut bacteria comprise Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus, Parabacteroides goldsteinii, or Prevotella tannerae.
 10. The pharmaceutical composition of claim 1, wherein the four to eleven strains of viable gut bacteria are in an amount are sufficient to treat or prevent a food allergy.
 11. The pharmaceutical composition of claim 1, wherein four to eleven strains of viable gut bacteria are present in substantially equal biomass.
 12. The pharmaceutical composition of claim 1, wherein four to eleven strains of viable gut bacteria are each present in an amount of at least about 1×108 CFUs/ml.
 13. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition does not comprise any of the Species Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Bilophila wadsworthia, Alistipes onderdonkii, Desulfovibrio species, Lactobacillus johnsonii, or Parasutterella excrementihominis.
 14. The pharmaceutical composition of claim 1, wherein minimal microbial consortium consisting essentially of four to eleven strains of viable gut bacteria is present in an amount sufficient to increase the proportion of T regulatory cells in a subject.
 15. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium comprises a viable, culturable, anaerobic gut bacterial strain that expresses exopolysaccharide, lipoteichoic acid (LTA), lipopolysaccharide (LPS) or other microbial adjuvant molecules that promote the development of regulatory T cells (Treg).
 16. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium comprises a viable, culturable, anaerobic gut bacterial strain that produces butyrate and/or propionate fermentation products via fermentation of carbohydrates and other carbon sources in the gut lumen.
 17. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium performs the full complement of bile acid transformations.
 18. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium comprises a viable, culturable, anaerobic gut bacterial strain that produces compounds capable of stimulating the aryl hydrocarbon receptor (AhR) receptor pathway in gut epithelial cells, antigen presenting cells and/or T cells to stimulate development of regulatory T cell responses.
 19. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium comprises a viable, culturable, anaerobic gut bacterial strain that produces compounds capable of stimulating the pregnane X receptor with beneficial effects upon gut barrier function and/or development of regulatory T cell responses.
 20. The pharmaceutical composition of claim 19, wherein the viable, culturable, anaerobic gut bacterial strain expresses desmolase or hydroxysteroid dehydrogenase.
 21. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium comprises a viable, culturable, anaerobic gut bacterial strain that produces compounds capable of stimulating the RORgamma (RAR-related orphan receptor gamma) pathways to stimulate development of regulatory T cell responses via direct stimulation or RORgamma-activated pathways in gut antigen presenting cells and/or epithelial cells that then stimulate regulatory T cell responses.
 22. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium comprises a viable, culturable, anaerobic gut bacterial strain that stimulates host production of mucins and complex glycoconjugates that improve gut barrier function and colonization by protective commensal species.
 23. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium comprises a viable, culturable, anaerobic gut bacterial strain that alters the gut luminal environment to reduce the deleterious activities of dysbiotic species promoting development of unhealthy allergic T cell responses to food antigens
 24. The pharmaceutical composition of claim 1, wherein the minimal microbial consortium comprises a viable, culturable, anaerobic gut bacterial strain that alters the gut luminal environment to promote improved colonization by other members of the administered consortium for any of the above stated effects, and/or colonization by existing beneficial species in the patients underlying microbiota.
 25. A method for increasing immune system tolerance, comprising administering the pharmaceutical composition of claim 1 to a subject in need thereof.
 26. The method of claim 25, wherein the pharmaceutical composition is formulated for oral delivery.
 27. The method of claim 25, administration of the pharmaceutical composition is in an amount sufficient to treat or prevent a food allergy. 